SCALABLE FERMENTATION PROCESS

Abstract

This invention provides a robust fermentation process for the expression of a capsid protein of a bacteriophage which is forming a VLP by self-assembly, wherein the process is scalable to a commercial production scale and wherein the expression rate of the capsid protein is controlled to obtain improved yield of soluble capsid protein. This is achieved by combining the advantages of fed-batch culture and of lactose induced expression systems with specific process parameters providing improved repression of the promoter during the growth phase and high plasmid retention throughout the process.

Description

FIELD OF THE INVENTION

[0001] This invention is related to the field of protein expression and fermentation technology. A process for the efficient expression of recombinant bacteriophage capsid protein in a bacterial host is described. The process leads to high yield of recombinant capsid protein which is capable of forming a virus-like particle (VLP) by self-assembly. Furthermore, the process is scalable from laboratory scale to fermenter volumes larger than 50 litres.

BACKGROUND OF THE INVENTION

[0002] Recent vaccination strategies make use of viruses or virus-like-particles (VLPs) to enhance the immune response towards antigens. For example, WO02/056905 demonstrates the utility of VLPs as a carrier to present antigens linked thereto in a highly ordered repetitive array. Such antigen arrays can cause a strong immune response, in particular antibody responses, against the linked antigen and are even capable of breaking the immune system's inherent tolerance towards self antigens. Such antigen arrays are therefore useful in the production of vaccines for the treatment of infectious diseases and allergies as well as for the efficient induction of self-specific immune responses, e.g. for the treatment of cancer, rheumatoid arthritis and various other diseases.

[0003] As indicated in WO02/056905 capsid proteins of bacteriophages are particularly suited as antigen carrier. They have been shown to efficiently self-assemble into VLPs upon expression in a bacterial host (Kastelein et al. 1983, Gene 23:245-254; Kozlovskaya et al. 1986, Dokl. Akad. Nauk SSSR 287:452-455). Moreover, capsid proteins of bacteriophages such as derived from fr (Pushko et al. 1993, Protein Engineering 6(8)883-891), Q.beta. (Kozlovska et al. 1993, Gene 137:133-137; Ciliens et al. 2000, FEBS Letters 24171:1-4; Vasiljeva et al 1998, FEBS Letters 431:7-11) and MS-2 (WO92/13081; Mastico et al. 1993, Journal of General Virology 74:541-548; Heal et al. 2000, Vaccine 18:251-258) have been produced in bacterial hosts using inducible promoters such as the trp promoter or a trp-T7 fusion (in the case of fr and Qb) or the tac promoter using IPTG as inducer substance (in the case of MS-2). The use of inducible promoters is beneficial, to avoid possible toxic effects of the recombinant capsid protein and the metabolic burden of protein expression which both might reduce the growth of the bacterial expression host and, ultimately, the yield of expressed protein.

[0004] However, the expression systems used so far for the expression of capsid proteins of bacteriophages have been applied in small scale fermentations, i.e. in laboratory scale and small batch cultures with volumes of typically clearly below 1 litre. An scale up of these systems comprising volumes of 50 litre and more is expected to diminish in a great extent the respective capsid protein yield due to increased promoter leakage and/or lowered plasmid retention.

[0005] A further problem associated with commercially desired high-level expression and rapid accumulation of recombinant capsid proteins of bacteriophages is the formation of incorrectly folded protein species and the formation of so called inclusion bodies, i.e. protein aggregates, which are insoluble and which may hamper further downstream processes. Thus, for bacteriophage MS-2 coat protein the formation of protein aggregates and of protein species which lost their ability to self-assemble to VLPs have been reported when the protein was expressed under the control of the strong T7 promoter after IPTG induction using the pET expression system (Peabody & Al-Bitar 2001, Nucleic Acid Research 29(22):e113).

[0006] High expression rates of the recombinant capsid protein may therefore have a negative impact on the yield of correctly assembled VLPs. The production of VLP-based vaccines in a commercial scale requires, therefore, the establishment of an efficient, and in particular scalable fermentation process for the expression of recombinant capsid protein of bacteriophages leading to a product of constant quality and purity having the capability of self-assembling into VLPs, whereby the formation of insoluble fractions of the capsid protein is minimised or avoided.

[0007] Therefore, it is an object of the present invention to provide a process for expression of a recombinant capsid protein of a bacteriophage which avoids or minimizes the disadvantage or disadvantages of the prior art processes, and in particular, which is scalable to a commercial scale and still leading to a product of constant quality and purity and the capability of self-assemblance to VLPs, and wherein the formation of insoluble fraction of the capsid protein is minimised or avoided.

SUMMARY OF THE INVENTION

[0008] The invention relates to a process for expression of a recombinant capsid protein of a bacteriophage, or a mutant or fragment thereof being capable of forming a VLP by self-assembly, said process comprising the steps of:.a) introducing an expression plasmid into a bacterial host, wherein said expression plasmid comprises an expression construct, wherein said expression construct comprises (i) a first nucleotide sequence encoding said recombinant capsid protein, or mutant or fragment thereof, and (ii) a promoter being inducible by lactose; b.) cultivating said bacterial host in a medium comprising a major carbon source; wherein said cultivating is performed in batch culture and under conditions under which said promoter is repressed by lad, wherein said lad is overexpressed by said bacterial host; c.) feeding said batch culture with said major carbon source; and d.) inducing said promoter with an inducer, wherein preferably said feeding of said batch culture with said major carbon source is continued.

[0009] This invention provides a robust fermentation process for the expression of a capsid protein of a bacteriophage which is forming a VLP by self-assembly, wherein the process is scalable to a commercial production scale and wherein the expression rate of the capsid protein leads to improved yield of soluble capsid protein. This is, in particular, achieved by improved repression of the promoter during the growth phase and high plasmid retention throughout the process. The expression system further avoids formation of insoluble protein aggregates by limiting the maximum expression rate occurring during the production phase.

[0010] In a preferred embodiment said bacteriophage is a RNA bacteriophage. More preferably, said RNA bacteriophage is selected from the group consisting of: a.) bacteriophage Q.beta.; b.) bacteriophage AP205; c.) bacteriophage fr; d.) bacteriophage GA; e.) bacteriophage SP; f.) bacteriophage MS2; g.) bacteriophage M11; h.) bacteriophage MX1; i.) bacteriophage NL95; j.) bacteriophage f2; k.) bacteriophage PP7 and 1.) bacteriophage R17. Preferably, said RNA bacteriophage is Q.beta.. More preferably said recombinant capsid protein comprises or alternatively consists of an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11. Still more preferably said recombinant capsid protein comprises SEQ ID NO:5, most preferably said recombinant capsid protein consists of SEQ ID NO:5.

[0011] In a further preferred embodiment said recombinant capsid protein comprises or alternatively consists of an amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. More preferably said recombinant capsid protein comprises SEQ ID NO:12, most preferably said recombinant capsid protein consists of SEQ ID NO:12.

[0012] In another embodiment of the present invention, said expression construct comprises a first stop codon, and wherein said first stop codon is TAA, and wherein preferably said TAA is located directly 3' of said first nucleotide sequence.

[0013] In a further embodiment said expression construct comprises a first stop codon and a second stop codon, wherein said first stop codon is located directly 3' of said first nucleotide sequence and wherein said second stop codon is located directly 3' of said first stop codon, and wherein at least one of said first or second stop codon is TAA.

[0014] In a further embodiment said expression construct comprises a first nucleotide sequence and a second nucleotide sequence, wherein said first nucleotide sequence is encoding a recombinant capsid protein, preferably Q.beta. CP, or a mutant or fragment thereof, and wherein said second nucleotide sequence is encoding any other protein, preferably the Q.beta. A1 protein or a mutant or fragment thereof, and wherein said first and said second nucleotide sequence are separated by exactly one sequence stretch comprising at least one TAA stop codon. In a preferred embodiment said expression construct comprises or alternatively consists of the nucleotide sequence of SEQ ID NO:6.

[0015] In a further embodiment said expression plasmid comprises or, more preferably, consists of the nucleotide sequence of SEQ ID NO:1.

[0016] In one embodiment of the invention said promoter is selected from the group consisting of the a.) tac promoter; b.) trc promoter; c.) tic promoter; d.) lac promoter; e.) lacUV5 promoter; f.) P.sub.syn promoter; g.) 1pp.sup.a promoter; h.) 1pp-lac romoter; i.) T7-lac promoter; j.) T3-lac promoter; k.) T5-lac promoter; and 1.) a promoter having at least 50% sequence homology to SEQ ID NO:2.

[0017] In a preferred embodiment said promoter has at least 50%, 60%, 70%, 80, 90, or 95%, preferably 98 to 100%, most preferably 99% sequence homology to SEQ ID NO:2. In a further preferred embodiment said promoter is selected from the group consisting of tic promoter, trc promoter and tac promoter. Even more preferably said promoter is the tac promoter. Most preferably said promoter comprises or alternatively consists of the nucleotide sequence of SEQ ID NO:2.

[0018] In one embodiment said major carbon source is glucose or glycerol, preferably glycerol.

[0019] In one embodiment said feeding of said batch culture is performed with a flow rate, wherein said flow rate increases with an exponential coefficient .mu., and wherein preferably said exponential coefficient .mu. is below .mu..sub.max.

[0020] In a further embodiment said inducing of said promoter is performed by co-feeding said batch culture with said inducer, preferably lactose and said major carbon source, preferably glycerol, at a constant flow rate.

[0021] In a further embodiment said inducing of said promoter is performed by co-feeding said batch culture with said inducer, preferably lactose and said major carbon source, preferably glycerol, at an increasing flow rate.

[0022] In a further embodiment said inducer is lactose, wherein preferably said lactose and said major carbon source are co-fed to said batch culture in a ratio of about 2:1 to 1:4 (w/w).

[0023] In a further embodiment said inducer is IPTG wherein preferably the concentration of said IPTG said medium is 0.001 to 5 mM, preferably 0.001 to 1 mM, more preferably 0.005 to 1 mM, still more preferably 0.005 to 0.5 mM. In a very preferred embodiment said concentration of IPTG is about 0.01 mM, most preferably 0.01 mM.

[0024] In one embodiment said lad is overexpressed by said bacterial host, wherein said overexpression is caused by lacI.sup.q or lacQ1, preferably by lacI.sup.q. In one embodiment said bacterial host comprises said lacI.sup.q gene or said lacQ1 gene, preferably said lacI.sup.q gene on its chromosome. In a further prefered embodiment said bacterial host comprises said lacI.sup.q gene or said lacQ1 gene, preferably said lacI.sup.q gene on a plasmid, preferably on a high copy number plasmid. In a further prefered embodiment said bacterial host comprises said lacI.sup.q gene or said lacQ1 gene, preferably said lacI.sup.q gene on said expression plasmid.

[0025] In one embodiment said bacterial host is selected from the group consisting of the strains E. coli RB791, E. coli DH2O and E. coli Y1088. Preferably said bacterial host is E. coli RB791.

[0026] In one embodiment said bacterial host comprises .beta.-galactosidase activity.

[0027] In one embodiment said cultivating and said feeding of said batch culture and said inducing of said promoter is performed at a temperature which is below the optimal growth temperature of said bacterial host. Preferably said temperature is between 23.degree. C. and 35.degree. C., more preferably between 25 and 33.degree. C., even more preferably between 27 and 32.degree. C., still more preferably between 28 and 31.degree. C. Even more preferably said temperature is about 30.degree. C., most preferably said temperature is 30.degree. C.

[0028] In one embodiment said cultivating and said feeding of said batch culture is performed at a temperature which is below the optimal growth temperature of said bacterial host, wherein preferably said temperature is between 23.degree. C. and 35.degree. C., more preferably between 25 and 33.degree. C., even more preferably between 27 and 32.degree. C., still more preferably between 28 and 31.degree. C., even more preferably said temperature is about 30.degree. C., most preferably said temperature is 30.degree. C., and said inducing of said promoter is performed at the optimal growth temperature of the bacterial host, preferably at about 37.degree. C.

[0029] In one embodiment said cultivating and said feeding of said batch culture and said inducing of said promoter is performed in the absence of an antibiotic.

[0030] In a specific embodiment said expression plasmid comprises or alternatively consists of the nucleotide sequence of SEQ ID NO:1, said major carbon source is glycerol, said feeding of said batch culture is performed with a flow rate, wherein said flow rate increases with an exponential coefficient .mu., and wherein said exponential coefficient .mu. is below .mu..sub.max, said inducing of said promoter by co-feeding said batch culture is performed with a constant flow rate, wherein lactose and glycerol are co-fed to the batch culture in a ratio of about 2:1 to about 1:4 (w/w), preferably about 1:1 to about 1:4 (w/w), most preferably about 1:3 (w/w), and wherein said cultivating and feeding of said batch culture and said inducing of said promoter is performed at a temperature between 27 and 32.degree. C., preferably about 30.degree. C., most preferably 30.degree. C.

[0031] In a further specific embodiment said expression plasmid comprises or alternatively consists of the nucleotide sequence of SEQ ID NO:30, said major carbon source is glycerol, said feeding of said batch culture is performed with a flow rate, wherein said flow rate increases with an exponential coefficient .mu., and wherein said exponential coefficient .mu. is below .mu..sub.max, said inducing of said promoter by co-feeding said batch culture is performed with a constant flow rate, wherein lactose and said major carbon source are co-fed to the batch culture in a ratio of about 2:1 to about 1:4 (w/w), preferably about 1:1 to about 1:4 (w/w), most preferably about 1:3 (w/w), and wherein said cultivating and feeding of said batch culture and said inducing of said promoter is performed at a temperature between 27 and 32.degree. C., preferably about 30.degree. C., most preferably 30.degree. C.

DESCRIPTION OF THE FIGURES

[0032] FIG. 1: Fermentation profile with pTac-nSD-Qb-mut (SEQ ID NO:1) in RB791 in 21 culture. Co-feeding during production phase was performed with medium containing 20% glycerol and 20% lactose. Shown are glycerol concentration [g/l] (circles); lactose concentration [g/l] (triangles); .beta.-Gal activity [U/ml*OD=1] (squares) and OD600 (diamonds) plotted against the process time [h].

DETAILED DESCRIPTION OF THE INVENTION

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0034] "about": within the meaning of the present application the expression about shall have the meaning of +/-10%. For example about 100 shall mean 90 to 110.

[0035] "promoter which is inducible by lactose" as used herein refers to a promoter which comprises regulatory elements of the lac operon. Such promoters are repressed by lad and can be induced by lactose or the synthetic inducer IPTG. The skilled person is aware that induction of a promoter by lactose requires .beta.-galactosidase activity in the bacterial host.

[0036] "located directly 3'": a nucleotide sequence N2 which is located directly 3' of another nucleotide sequence N1 refers to a continuous sequence having the conformation 5'-N1-N2-3' wherein N1 and N2 are directly connected and not separated by additional sequence elements.

[0037] "sequence stretch": as used herein the term "sequence stretch" refers to a continuous nucleotide sequence which consists of less than 50, preferably less than 20, more preferably less than 10, even more preferably less than 5 nucleotides. In a further preferred embodiment the sequence stretch comprises or alternatively consists of at least one, preferably one, TAA stop codon. In another embodiment the sequence stretch comprises or alternatively consists of at least one, preferably one, TAA and at least one, preferably one, TGA stop codon. In further preferred embodiment the sequence stretch comprises or alternatively consists of SEQ ID NO:32.

[0038] "bacterial host": as used herein the term "bacterial host" refers to a bacterial organism which is hosting or capable of hosting an expression plasmid of the invention, wherein "hosting" involves the replication of the expression plasmid and maintenance of the expression plasmid during cell division.

[0039] "culture": in the context of the instant invention a "culture" comprises a bacterial host in a medium ("bacterial culture"), wherein typically said medium is supporting the growth of said bacterial host.

[0040] "batch culture" as used herein relates to a culture, i.e. a bacterial host in a medium, wherein said culture constitutes a closed system, i.e. typically and preferably no addition or removal of medium takes place during the cultivation time. Therefore, in contrast to a continuous culture, typically and preferably the density of the bacterial host in the batch culture continuously increases with progressing cultivation time. Batch culture does not exclude the addition of compounds required for the control of the process, such as, for example, inducer, oxygen, and alkali or acid to control the pH.

[0041] "fed batch culture": as used herein is a culture which is supplied with additional medium comprising a substrate, preferably the major carbon source of the bacterial host (feed or co-feed medium). In the context of the application this process is referred to by the terms "feeding said batch culture" (medium comprises the major carbon source) and "co-feeding said batch culture" (medium comprises the major carbon source and the inducer, preferably lactose). Typically and preferably, no removal of medium except for analytical purposes takes place during cultivation time of a fed batch culture.

[0042] "Preculture": a culture, preferably a batch culture, which is used to produce the inoculum for a culture of a larger volume, e.g. the culture in which the recombinant capsid protein is produced (production culture). A preculture can be performed in two or more steps, wherein a second preculture is inoculated with a first preculture etc. to produce a sufficiently large inoculum for the production culture. The first and/or subsequent precultures may comprise an antibiotic to improve plasmid stability.

[0043] "substrate": as used herein refers to a compound in the culture medium which contributes to the carbon and energy supply of the bacterial host. The terms "substrate" therefore encompasses any compound contained in the medium contributing to the carbon supply of the bacterial host. Typical substrates for bacteria are sugar, starch, glycerol, acetate and any other organic compound which can be metabolized by bacteria. Therefore, the term "substrate" includes the major carbon source but also, for example, lactose.

[0044] "Major carbon source" as used herein refers to the compound in the culture medium which contributes most to the carbon and energy supply of the bacterial host during the growth phase. The major carbon source thus is the major substrate of the bacterial host. The major carbon source is typically a sugar such as sucrose or glucose, or glycerol, and preferably glucose or glycerol. Though lactose could in principal act as a major carbon source for a bacterial host, in the context of the instant invention the term "major carbon source" typically and preferably does not include lactose.

[0045] Phases of the process of the invention: The process of the invention is characterised by different phases which refer to different physiological conditions of the bacterial host with respect to its growth and the repression/induction status of the expression construct.

[0046] "Growth phase": The growth phase is initiated by said cultivating said bacterial host in a medium. The growth phase is preferably characterized by conditions under which the promoter driving the expression of the recombinant capsid protein is repressed and the growth phase is terminated with said inducing said promoter with an inducer. The growth phase can be further divided in a "batch phase" and a "feed phase". Said batch phase is initiated by said cultivating said bacterial host in a medium. The batch phase comprised a "lag phase" during which the bacterial host is not yet growing or growing with a non-exponential rate, typically and preferably a linear rate. The growth phase further comprises an "exponential growth phase" which directly follows the lag phase. No feeding of said culture takes place during the batch phase, thus the exponential growth phase is terminated by the consumption of the substrate by the bacterial host. The growth phase further comprises a "feed phase" which is directly following the batch phase and which is initiated by said feeding of said batch culture with said major carbon source. The feed phase is characterised by a growth rate of the bacterial host which is directly dependent on the flow rate of the feed medium containing the major carbon source.

[0047] "production phase": The growth phase is followed by the production phase which is initiated by said inducing said promoter with an inducer, wherein typically and preferably said feeding of said batch culture with said major carbon source is continued.

[0048] "Conditions under which the promoter is repressed": it is to be understood that the repression of a promoter is an equilibrium of formation and dissociation of the repressor-operator complex and that even stringently repressed promoters may show a certain expression rate also in the absence of their inducer. Therefore, as used within the application the term "conditions under which the promoter is repressed" relates to conditions, wherein at the end of the growth phase, i.e. directly before the addition of inducer to the culture, the recombinant capsid protein is expressed to a level which does not exceed a concentration in the medium of 200 mg/l, preferably 150 mg/l, more preferably 100 mg/l, as determined by the HLPC method of Example 17. Most preferably, the concentration of the recombinant protein is below the detection level of said method.

[0049] "Inducer": within the meaning of the in invention the term "inducer" relates to any substance which directly or indirectly interacts with an inducible promoter and thereby facilitates expression from said promoter; for example, inducers of "a promoter inducible by lactose", such as the lac or tac promoter, are IPTG, lactose and allolactose.

[0050] "Coat protein"/"capsid protein": The term "coat protein" and the interchangeably used term "capsid protein" within this application, refers to a viral protein, preferably a subunit of a natural capsid of a virus, preferably of a RNA bacteriophage, which is capable of being incorporated into a virus capsid or a VLP. For example, the specific gene product of the coat protein gene of RNA bacteriophage Q.beta. is referred to as "Q.beta. CP", whereas the "coat proteins" or "capsid proteins" of bacteriophage Q.beta. comprise the "Q.beta. CP" as well as the Al protein.

[0051] "Recombinant capsid protein": A capsid protein which is synthesised by a recombinant host cell. "Polypeptide": As used herein the term "polypeptide" refers to a polymer composed of amino acid residues, generally natural amino acid residues, linked together through peptide bonds. Although a polypeptide may not necessarily be limited in size, the term polypeptide is often used in conjunction with peptide of a size of about ten to about 50 amino acids.

[0052] "Protein": As used herein, the term protein refers to a polypeptide generally of a size of above 20, more particularly of above 50 amino acid residues. Proteins generally have a defined three dimensional structure although they do not necessarily need to, and are often referred to as folded, in opposition to peptides and polypeptides which often do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded.

[0053] "Recombinant host cell": As used herein, the term "recombinant host cell" refers to a host cell into which one ore more nucleic acid molecules of the invention have been introduced.

[0054] "Recombinant VLP": The term "recombinant VLP", as used herein, refers to a VLP that is obtained by a process which comprises at least one step of recombinant DNA technology. The term "VLP recombinantly produced", as used herein, refers to a VLP that is obtained by a process which comprises at least one step of recombinant DNA technology. Thus, the terms "recombinant VLP" and "VLP recombinantly produced" are interchangeably used herein and should have the identical meaning.

[0055] "RNA-bacteriophage": As used herein, the term "RNA-bacteriophage" refers to RNA viruses infecting bacteria, preferably to single-stranded positive-sense RNA viruses infecting bacteria.

[0056] "Virus-like particle (VLP)": as used herein, the term "virus-like particle" refers to a structure resembling a virus particle or it refers to a non-replicative or non-infectious, preferably a non-replicative and non-infectious virus particle, or it refers to a non-replicative or non-infectious, preferably a non-replicative and non-infectious structure resembling a virus particle, preferably a capsid of a virus. The term "non-replicative", as used herein, refers to being incapable of replicating the genome comprised by the VLP. The term "non-infectious", as used herein, refers to being incapable of entering the host cell. Preferably a virus-like particle in accordance with the invention is non-replicative and/or non-infectious since it lacks all or part of the viral genome or genome function. Typically a virus-like particle lacks all or part of the replicative and infectious components of the viral genome. A virus-like particle in accordance with the invention may contain nucleic acid distinct from their genome. A typical and preferred embodiment of a virus-like particle in accordance with the present invention is a viral capsid such as the viral capsid of the corresponding virus, bacteriophage, preferably RNA-phage. The terms "viral capsid" or "capsid", refer to a macromolecular assembly composed of viral protein subunits. Typically, there are 60, 120, 180, 240, 300, 360 and more than 360 viral protein subunits. Typically and preferably, the interactions of these subunits lead to the formation of viral capsid or viral-capsid like structure with an inherent repetitive organization, wherein said structure is, typically, spherical or tubular. For example, the capsids of RNA bacteriophages or HBcAgs have a spherical form of icosahedral symmetry.

[0057] "Virus-like particle of a RNA bacteriophage": As used herein, the term "virus-like particle of a RNA bacteriophage" refers to a virus-like particle comprising, or preferably consisting essentially of or consisting of coat proteins, mutants or fragments thereof, of a RNA bacteriophage. In addition, virus-like particle of a RNA bacteriophage resembling the structure of a RNA bacteriophage, being non replicative and/or non-infectious, and lacking at least the gene or genes encoding for the replication machinery of the RNA bacteriophage, and typically also lacking the gene or genes encoding the protein or proteins responsible for viral attachment to or entry into the host. Preferred VLPs derived from RNA bacteriophages exhibit icosahedral symmetry and consist of 180 subunits. A preferred method to render a virus-like particle of a RNA bacteriophage non replicative and/or non-infectious is by genetic manipulation.

[0058] one, a, or an: When the terms "one," "a," or "an" are used in this disclosure, they mean "at least one" or "one or more," unless otherwise indicated.

[0059] "Sequence identity": The amino acid sequence identity of polypeptides can be determined conventionally using known computer programs such as the Bestfit program. When using Bestfit or any other sequence alignment program, preferably using Bestfit, to determine whether a particular sequence is, for instance, 95% identical to a reference amino acid sequence, the parameters are set such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed. This aforementioned method in determining the percentage of identity between polypeptides is applicable to all proteins, polypeptides or a fragment thereof disclosed in this invention.

[0060] "Sequence homology": The homology of nucleotide sequences can for example be determined by the program blastn which is an implementation of the BLAST algorithm, preferably using the default settings of the software.

[0061] "Fragment of a protein", in particular fragment of a recombinant protein or recombinant coat protein, as used herein, is defined as a polypeptide, which is of at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% the length of the wild-type recombinant protein, or coat protein, respectively and which preferably retains the capability of forming VLP Preferably the fragment is obtained by at least one internal deletion, at least one truncation or at least one combination thereof. Further preferably the fragment is obtained by at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3 or at most 2 internal deletions; by at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3 or at most 2 truncations; or by at most 3, preferably at most 2, most preferably by exactly one combination thereof. Most preferably the fragment is obtained by exactly one internal deletion, exactly one truncation or by a combination thereof.

[0062] The term "fragment of a recombinant protein" or "fragment of a coat protein" shall further encompass polypeptide, which has at least 80%, preferably 90%, even more preferably 95% amino acid sequence identity with the "fragment of a recombinant protein" or "fragment of a coat protein", respectively, as defined above and which is preferably capable of assembling into a virus-like particle.

[0063] The term "mutant recombinant protein" or the term "mutant of a recombinant protein" as interchangeably used in this invention, or the term "mutant coat protein" or the term "mutant of a coat protein", as interchangeably used in this invention, refers to a polypeptide having an amino acid sequence derived from the wild type recombinant protein, or coat protein, respectively, wherein the amino acid sequence is at least 80%, preferably at least 85%, 90%, 95%, 97%, or 99% identical to the wild type sequence and preferably retains the ability to assemble into a VLP.

[0064] The invention is related to an efficient fermentation process for the production of a VLP of a bacteriophage. The process is improved with respect to yield of the VLP and can be scaled up to a commercial production scale. The process encompasses the expression of recombinant capsid protein of bacteriophages in a bacterial host under conditions which allow the capsid protein to self-assemble into VLPs spontaneously.

[0065] Specific examples of VLPs which can be produced by the process of the invention are VLPs of bacteriophages, preferably RNA bacteriophages. In one preferred embodiment of the invention, the virus-like particle of the invention comprises, consists essentially of, or alternatively consists of, recombinant coat proteins, mutants or fragments thereof, of a RNA-phage. Preferably, the RNA-phage is selected from the group consisting of a) bacteriophage Q.beta.; b) bacteriophage R17; c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g) bacteriophage M11; h) bacteriophage MX1; i) bacteriophage NL95; k) bacteriophage f2; 1) bacteriophage PP7 and m) bacteriophage AP205.

[0066] In one preferred embodiment of the invention, VLPs are produced comprising coat protein, mutants or fragments thereof, of RNA bacteriophages, wherein the coat protein has an amino acid sequence selected from the group consisting of: (a) SEQ ID NO:5 referring to Q.beta. CP; (b) a mixture of SEQ ID NO:5 and SEQ ID NO:15 (Q.beta. A1 protein); (c) SEQ ID NO:16 (R17 capsid protein); (d) SEQ ID NO:17 (fr capsid protein); (e) SEQ ID NO:18 (GA capsid protein); (f) SEQ

[0067] ID NO:19 (SP capsid protein); (g) a mixture of SEQ ID NO:19 and SEQ ID NO:20; (h) SEQ ID NO:21 (MS2 capsid protein); (i) SEQ ID NO:22 (M11 capsid protein); (j) SEQ ID NO:23 (MX1 capsid protein); (k) SEQ ID NO:24 (NL95 capsid protein); (1) SEQ ID NO:25 (f2 capsid protein); (m) SEQ ID NO:26 (PP7 capsid protein); and (n) SEQ ID NO:12 (AP205 capsid protein).

[0068] Upon expression in E. coli, the N-terminal methionine of Q.beta. coat protein is usually removed (Stoll, E. et al., J. Biol. Chem. 252:990-993 (1977)). VLP composed of Q.beta. coat proteins where the N-terminal methionine has not been removed, or VLPs comprising a mixture of Q.beta. coat proteins where the N-terminal methionine is either cleaved or present are also within the scope of the present invention.

[0069] In one preferred embodiment of the invention, the VLP is a mosaic VLP comprising or alternatively consisting of more than one amino acid sequence, preferably two amino acid sequences, of coat proteins, mutants or fragments thereof, of a RNA bacteriophage.

[0070] In one very preferred embodiment, the VLP comprises or alternatively consists of two different coat proteins of a RNA bacteriophage, said two coat proteins have an amino acid sequence of SEQ ID NO: 5 and SEQ ID NO:15, or of SEQ ID NO:19 and SEQ ID NO:20.

[0071] In preferred embodiments of the present invention, the produced VLP comprises, or alternatively consists essentially of, or alternatively consists of recombinant coat proteins, mutants or fragments thereof, of the RNA-bacteriophage Q.beta., fr, AP205 or GA.

[0072] In one preferred embodiment, the VLP is a VLP of RNA-phage Q.beta.. The capsid or virus-like particle of Q.beta. shows an icosahedral phage-like capsid structure with a diameter of 25 nm and T=3 quasi symmetry. The capsid contains 180 copies of the coat protein, which are linked in covalent pentamers and hexamers by disulfide bridges (Golmohammadi, R. et al., Structure 4:543-5554 (1996)).

[0073] Preferred virus-like particles of RNA bacteriophages, in particular of Q.beta. and fr in accordance of this invention are disclosed in WO 02/056905, the disclosure of which is herewith incorporated by reference in its entirety. Particular Example 18 of WO 02/056905 gave detailed description of preparation of VLP particles from Q.beta..

[0074] In another preferred embodiment, the VLP is a VLP of RNA bacteriophage AP205. Assembly-competent mutant forms of AP205 VLPs, including AP205 coat protein with the substitution of proline at amino acid 5 to threonine, may also be used in the practice of the invention and leads to other preferred embodiments of the invention. WO 2004/007538 describes, in particular in Example 1 and Example 2, how to obtain VLP comprising AP205 coat proteins, and hereby in particular the expression and the purification thereto. WO 2004/007538 is incorporated herein by way of reference.

[0075] In one preferred embodiment, the VLP comprises or consists of a mutant coat protein of a virus, preferably a RNA bacteriophage, wherein the mutant coat protein has been modified by removal of at least one lysine residue by way of substitution and/or by way of deletion. In another preferred embodiment, the VLP of the invention comprises or consists of a mutant coat protein of a virus, preferably a RNA bacteriophage, wherein the mutant coat protein has been modified by addition of at least one lysine residue by way of substitution and/or by way of insertion. The deletion, substitution or addition of at least one lysine residue allows varying the degree of coupling with an antigen.

[0076] VLPs or capsids of Q.beta. l coat protein display a defined number of lysine residues on their surface, with a defined topology with three lysine residues pointing towards the interior of the capsid and interacting with the RNA, and four other lysine residues exposed to the exterior of the capsid.

[0077] Q.beta. mutants, of which exposed lysine residues are replaced by arginines are also encompassed by the present invention. Preferably these mutant coat proteins comprise or alternatively consist of an amino acid sequence selected from the group of a) Q.beta.-240 (SEQ ID NO:7, Lys13.fwdarw.Arg); b) Q.beta.-243 (SEQ ID NO:8, Asn10.fwdarw.Lys); c) Q.beta.-250 (SEQ ID NO:9, Lys2.fwdarw.Arg); d) Q.beta.-251 (SEQ ID NO:10, Lys16.fwdarw.Arg); and e) Q.beta.-259 (SEQ ID NO:11, Lys2.fwdarw.Arg, Lys16.fwdarw.Arg). The construction, expression and purification of the above indicated Q.beta. mutant coat proteins, mutant Q.beta. coat protein VLPs and capsids, respectively, are described in WO02/056905. In particular is hereby referred to Example 18 of above mentioned application.

[0078] In a further preferred embodiment the recombinant capsid protein is a capsid protein of bacteriophage AP205 having the amino acid sequence depicted in SEQ ID NO:12 or a mutation thereof, which is capable of forming a VLP, for example the proteins AP205P5T (SEQ ID NO:13) or AP205 N14D (SEQ ID NO:14.).

[0079] In a very preferred embodiment said recombinant capsid protein is composed of the 133 amino acid coat protein C of E. coli RNA bacteriophage Q.beta. comprising or preferably consisting of the amino acid sequence depicted in SEQ ID NO:5, wherein preferably said recombinant capsid protein is capable of forming a VLP by self-assembly.

[0080] In one embodiment, the expression construct comprises a first stop codon and a second stop codon, wherein said first stop codon is located directly 3' of said first nucleotide sequence and wherein said second stop codon is located directly 3' of said first stop codon, and wherein at least one of said first or second stop codon is TAA. For example, plasmid pTac-nSDAP205 (SEQ ID NO:30) comprises the naturally occurring TAA stop codon as a first stop codon and an additional TGA stop codon directly 3' of the first stop codon.

[0081] In a preferred embodiment the expression construct comprises a first nucleotide sequence and a second nucleotide sequence, wherein said first nucleotide sequence is encoding a recombinant capsid protein, preferably Q.beta. CP, or a mutant or fragment thereof, most preferably SEQ ID NO:5, and wherein said second nucleotide sequence is encoding any other protein, preferably the Q.beta. A1 protein or a mutant or fragment thereof, most preferably SEQ ID NO:15, and wherein said first and said second nucleotide sequence are separated by exactly one sequence stretch comprising at least one TAA stop codon. In one embodiment said TAA stop codon is generated by replacing the naturally occurring stop codon, preferably TGA by the sequence TAA. Alternatively and more preferably said TAA stop codon is generated by replacing the naturally occurring stop codon, preferably TGA by the sequence TAATGA (SEQ ID NO:32).

[0082] For example, the region of Q 2 gene C corresponds to the NCBI GenBank Acc. No. M99039 (nucleotides 46-1062). Gene C contains a first nucleotide sequence encoding the 133-amino acid Q.beta. coat protein (SEQ ID NO:5) and a second nucleotide sequence encoding the 329-amino acid read through protein A1 (SEQ ID NO:15). Nucleotides 1-399 of SEQ ID NO:6 (nucleotides 46-444 of NCBI GenBank Acc. No. M99039) correspond to said first nucleotide sequence encoding the 133-amino acid Q.beta. CP, Nucleotides 400 to 402 of SEQ ID NO:6 correspond to the strong TAA stop codon and nucleotides 403 to 405 of SEQ ID NO:6 to the leaky TGA stop codon, which is followed by said second nucleotide sequence (Q.beta. A1). Surprisingly, it was found that the presence of the nucleotide sequence relating to A1 in the expression construct results in higher RNA stability and, thus, in improved yield of Q.beta. CP and VLP as compared to a construct wherein the A1 sequence is deleted.

[0083] The expression of a recombinant protein can significantly reduce the growth rate of the bacterial host due to toxic effects of the accumulating protein and due to the metabolic burden caused by the protein synthesis. In particular cell lysis and low plasmid retention may occur. Inducible promoters provide for the possibility to separate the growth phase from the production phase of a fermentation process. Inducible promoters are repressed by a repressor molecule during the growth phase of the bacterial host and are induced by exposing the bacterial host to inductive conditions during the production phase. Inducible promoters therefore allow the bacterial host to grow fast, preferably exponentially during the growth phase and to reach high cell densities. Thus, inducible promoters provide for high yield of the expression product at the end of the production phase. Therefore, the usage of inducible promoters for the expression of recombinant protein is preferred.

[0084] A well known example for an inducible promoter is the lac promoter which forms part of the lac operon and which can be induced by addition of lactose or the strong synthetic inducer isopropylthio-.beta.-D-galactosid (IPTG) to the growth medium of the bacterial host. Donavan et al. 2000 (Can. J. Microbiol 46:532-541) report on an improved process for the expression of a monoclonal antibody fragment under the control of the lac promoter. Further examples of inducible promoters are provided in table 1 of Makrides 1996 (Microbiological Reviews, p. 512-538).

[0085] A typical drawback of expression systems based on inducible promoters is the "leakiness" of the promoter, meaning that the promoter is only insufficiently repressed and causes a certain expression rate of the recombinant protein during the growth phase. This typically leads to a reduced cell density or to plasmid instability and, as a consequence, to reduced yield of the recombinant protein Makrides 1996 (Microbiological Reviews, p. 512-538). An example of a promoter which is prone to insufficient repression is the VHb promoter which is repressed under high oxygen conditions and induced upon oxygen depletion.

[0086] For the purpose of the invention promoters are preferred which are stringently repressed. In one embodiment the promoter is repressed by the repressor lacI. Examples of such promoters are disclosed in Makrides 1996 (Microbiol. Rev. 60:512-538), Goldstein & Doi 1995 (Biotechnology Annual Review 1:105-128), Hannig & Makrides 1998 (TIBTECH 16:54-60) and Stevens 2000 (Structures 8, R177-R185). In a preferred embodiment the promoter is inducible by lactose, more preferably it is selected from the group consisting of lac, lacUV5, tac, trc, P.sub.syn lpp.sup.a, lpp-lac, T7-lac, T3-lac, and T5-lac. Especially preferred for the purpose of the invention is the tac promoter (SEQ ID NO:2) or a mutation or variant thereof. Within the scope of the invention are mutants or truncated or deleted variants of the tac promoter having a sequence homology with SEQ ID NO:2 which is at least 50%, 60%, 70%, 80, 90, or 95%, preferably 98 to 100%, most preferably 99%. Wherein the promoter strength of such mutated truncated or deleted variant is comparable to that of the promoter of SEQ ID NO:2. The skilled person will be able to determine the promoter strength of a given sequence by comparative expression studies using standard methods. In a specific embodiment of the invention the promoter driving the expression of the recombinant capsid protein comprises or alternatively consists of SEQ ID NO:2. The tac promoter is a fusion product of the -10 region of the lacUV5 promoter and the -35 region of the trp promoter and combines the high transcription efficiency of trp with the regulatory elements of the lac promoter (de Boer et al. 1983, PNAS 80:21-25; Amann et al. 1983 Gene 25:167-178). It provides for sufficiently high expression rates and high protein yield while avoiding the formation of insoluble or incorrectly folded recombinant protein which may occur with stronger promoters, such as the T7 promoter. The trc and the tic promoter are mutated versions of the tac promoter (Brosius et al. 1985, The Journal of Biological Chemistry 260(6):3539-3541). In a further preferred embodiment the promoter is selected from the group consisting of tic, trc and tac.

[0087] For the construction of an expression construct for the purpose of the invention the promoter is operably linked to said first nucleotide sequence encoding the recombinant capsid protein via a ribosome binding site (Shine-Dalgarno sequence, SD), typically comprising an

[0088] ATG start codon at its 3' end. Suitable Shine-Dalgarno sequences for the purpose of the invention are well known in the art (Dalboge et al. 1988, DNA 7(6):399-405; Ringquist et al. 1992, Mol. Micr. 6:1219-1229). In one embodiment of the invention the expression construct comprises the SD sequence of Dalboge et al. 1988 (DNA 7(6):399-405) which is depicted in SEQ ID NO:4. In another, preferred, embodiment the expression construct comprises a Shine-Dalgamo sequence of Ringquist et al. 1992 (Mol. Micr. 6:1219-1229, SEQ ID NO:3, nSD). Surprisingly, it was found that SEQ ID NO:3 is particularly suited for the purpose of the invention because it results in improved expression levels and improved yield of recombinant capsid protein. SEQ ID NO:3 is especially suited to enhance the expression of AP205 capsid protein. In a preferred embodiment of the invention the expression construct comprises a Shine-Dalgamo sequence selected form the group consisting of SEQ ID NO:3 and SEQ ID NO:4, preferably said Shine-Dalgarno sequence is SEQ ID NO:3.

[0089] Transcriptional terminators are functional elements of expression constructs. The skilled person will be able to choose a suitable terminator sequence form a wide range of sources. In a preferred embodiment of the invention said expression construct comprises a terminator sequence, wherein preferably said terminator sequence is operably linked to said first nucleotide sequence, wherein further preferably said terminator sequence is the rRNB terminator sequence, most preferably SEQ ID NO:28.

[0090] For the purpose of plasmid selection the skilled person will typically use an antibiotic resistance marker gene. Examples of antibiotic resistance genes which are widely used in the art and which are suitable for the purpose of the invention are resistance genes against the antibiotics ampicillin, tetracyclin and kanamycin. The use of kanamycin as a selective agent in the frame of a process for the production of a VLP is generally preferred because of the lower allergenic potential of kanamycin as compared to alternative antibiotics and because of the lower safety concerns resulting thereof for the use of the VLP as a vaccine. Furthermore, kanamycin provides better plasmid retention as compared to alternative antibiotics such as ampicillin. The kanamycin 3'-phosphotransferase gene (SEQ ID NO:29) which is derived from the transposon Tn903 is therefore a particularly useful selectable marker gene.

[0091] The addition of antibiotics to the medium is generally undesirable in a commercial production process for cost and safety reasons. In the context of the invention antibiotics, preferably kanamycin, are typically and preferably used for the selection of the expression strain. Media used in the production process are essentially free of antibiotics, in particular kanamycin. However, addition of an antibiotic to precultures used to produce the inoculum for the production culture can improve plasmid retention throughout the process (Example 10).

[0092] The skilled person will create expression plasmids comprising expression constructs which are useful for the production of VLPs of bacteriophages by combining the genetic elements described above applying standard methods of molecular biology. Particularly useful expression plasmids for the purpose of the invention are pTac-nSDQb-mut (SEQ ID NO:1) for the production of Q.beta. VLP and pTac-nSDAP205 (SEQ ID NO:30) for the production of AP205 VLP. The construction of these specific expression plasmids is described in detail in the Examples section.

[0093] The expression plasmids are transformed to a bacterial expression host by any method known in the art, preferably by electroporation. Individual clones of the host comprising the expression plasmid are selected for maximal expression of the recombinant capsid protein by SDS-PAGE after cell lysis. Selected clones of the expression host comprising the expression plasmid can be stored as frozen glycerol cultures.

[0094] Said bacterial host can be chosen from any bacterial strain capable of replicating and maintaining said expression plasmid during cell division. Preferred bacterial hosts are Escherichia coli strains having the specific features described in the following sections.

[0095] The repression of the promoter is improved by overexpression of the repressor by the bacterial host. In one embodiment said cultivating of said bacterial host is performed in batch culture and under conditions under which said promoter is repressed by lad. In a preferred embodiment the gene causing overexpression of said lad in said bacterial host is located on a plasmid, preferably on said expression plasmid. Alternatively, said gene is located on a separate plasmid contained in said bacterial host, wherein said separate plasmid preferably is a high copy number plasmid. Alternatively, and most preferably said gene is located on the chromosome of said bacterial host.

[0096] One example of a gene causing overexpression of lacI is lacI.sup.q (Menzella et al. 2003, Biotechnology and Bioengineering 82(7)809-817) which is a single CG to TA change at -35 of the promoter region of lacI which causes a 10 fold increase in LacI expression. A further example is lacIQ1 (Glascock & Weickert 1998, Gene 223(1-2):221-231). Improved repression of the promoter during the growth phase results in improved plasmid retention and higher cell density and, ultimately, in improved protein yield. For example, bacterial strains comprising the lacIq gene overexpress the lad repressor molecule and therefore prevent formation of the recombinant protein during the growth phase more efficiently than strains comprising the wildtype gene. In a preferred embodiment the gene causing overexpression of said lad is lacIQ1 or lacI.sup.q, preferably lacI.sup.q. In a specifically preferred embodiment said bacterial host comprises the lacI.sup.q gene on its chromosome.

[0097] In one embodiment said inducing of said promoter is performed with an inducer, wherein said inducer is preferably selected from IPTG and lactose, most preferably said inducer is lactose. Upon exposure of the bacterial host to an inducer, the repressor is inactivated and the promoter becomes active. Addition of the strong inducer IPTG to the culture medium results in an immediate increase of the expression rate of the recombinant protein to a high level because IPTG directly enters the cells by diffusion and binds and inactivates the active repressor lad. Inactivated lacI repressor molecules dissociates from the operator and allow high level transcription from the promoter. IPTG is not metabolized by the cell and the transcription continues with high rates until other metabolic parameters become limiting.

[0098] As mentioned before, high expression rates may lead to the formation of insoluble recombinant protein which is not capable of forming a VLP by self-assembly. Induction of protein expression with high concentrations of IPTG is particularly prone to the formation of insoluble protein. Therefore, induction of the promoter is preferably achieved by the addition of IPTG in concentrations which are below the concentration which causes the expression to occur at its maximum rate (Kopetzki et al. 1989, Mol Gen Genet 216:149-155).

[0099] In a preferred embodiment said inducing of said promoter is performed with IPTG, wherein the concentration of said IPTG in said medium is about 0.001 to 5 mM, preferably 0.001 to 1 mM, more preferably 0.005 to 1 mM, still more preferably 0.005 to 0.5 mM. In a specifically preferred embodiment the concentration of said IPTG is about 0.01 mM, most preferably 0.01 mM.

[0100] Alternatively, induction of the promoter is achieved by the addition of lactose. Induction of recombinant protein expression with lactose requires that the bacterial host is capable of taking up lactose from the medium, e.g. by Lac permease and that it comprises .beta.-galactosidase activity. The Lac permease dependent uptake of lactose into the cells follows a slower kinetic than the uptake of IPTG by diffusion. Furthermore, lactose does not directly interact with the lac operon but is converted by .beta.-galactosidase to allolactose (1-6-O-.beta.-galactopyranosyl-D-glucose) which is the actual inducer of the promoter. Induction of recombinant protein expression by the addition of lactose is advantageous because it avoids the immediate increase of the expression rate to a maximum level upon addition of the inducer and, thus, it reduces the risk of the formation of insoluble protein.

[0101] Allolactose is metabolised by the bacterial host during the production phase and contributes carbon and metabolic energy to the bacterial metabolism. This may further contribute to improved protein yield as compared to induction with IPTG. Furthermore, induction by lactose allows to a certain extend the control of the expression rate of the recombinant protein during the production phase via the lactose concentration in the medium. Induction by lactose is further preferred in a pharmaceutical production process because IPTG is expensive and is believed to be toxic. Its removal needs to be demonstrated at the end of a the production process.

[0102] In a preferred embodiment said inducing of said promoter is performed by the addition of lactose to said batch culture, wherein preferably said bacterial host is capable of taking up lactose from the medium and wherein further preferably said bacterial host comprises .beta.-galactosidase activity. Such bacteria strains can, for example, be obtained from strain collections such as ATTC (http://www.atcc.org). In a preferred embodiment, said bacterial host is an E. coli strain, preferably an E. coli strain selected from the group consisting of RB791, DH2O, Y1088, W3110 and MG1655. Most preferably, said bacterial host is E. coli RB791. In a still more preferred embodiment said promoter is the tac promoter or mutant or variant thereof and said bacterial host is an E. coli strain which further comprises a gene causing overexpression of a repressor of the tac promoter, wherein said gene preferably is lacI.sup.q. The pH of the culture medium of the bacterial host can be controlled during the fermentation process and regulated by the addition of acidic or alkaline solutions using methods which are well known in the art. In one embodiment, said cultivating of said bacterial host and said feeding of said batch culture is performed under conditions, wherein the pH of the medium is controlled. In a preferred embodiment said pH is between 5.5 and 8.0, more preferably between 6.5 and 7.5, even more preferably between 6.7 and 7.0 and most preferably said pH of said medium is 6.8. Said pH of said medium may be kept constant during the process or it may follow a certain profile during the different phases of the process within the pH ranges specified above. In a preferred embodiment said pH is kept constant at a value of 6.7 to 7.0, preferably said pH it is kept constant at 6.8.

[0103] The process of the invention comprises a growth phase, wherein said growth phase comprises a batch and a feed phase, wherein said growth phase and simultaneously said batch phase are initiated by said cultivating said bacterial host and wherein said feed phase is initiated by said feeding of said batch culture with said major carbon source.

[0104] The oxidative capacity of bacteria cells is limited and high concentrations of the substrate may cause the formation of reduced products like acetate, which may lead to undesired acidification of the medium and to reduced growth of the bacteria. Therefore, the bacterial host is grown in a fed-batch culture on a minimum medium with a limited quantity of substrate. In one embodiment said cultivating of said bacterial host is performed in a medium comprising said major carbon source, wherein said medium preferably is a minimal medium, preferably a chemically defined minimal medium. Most preferably said medium is R27 medium as described in Example 5.

[0105] At the end of the batch phase, when the substrate contained in the medium is almost exhausted, medium containing the major carbon source (feed medium) is fed to said batch culture at the same rate as the desired growth rate of the bacterial host, i.e. the growth rate of the bacterial host is limited by the feed rate of the substrate. It is understood by the skilled person that the decisive parameter is the actual mass flow of the substrate, preferably the major carbon source, and other nutrients required to maintain growth. Since in practice a constant composition of the feed medium can be assumed, the flow rate refers to the volume flow of the medium. The same consideration applies to the co-feed medium (see below).

[0106] Therefore, in one embodiment said feeding of said batch culture with said major carbon source is performed with a flow rate, wherein said flow rate is limiting the growth rate of said bacterial host.

[0107] During the feed phase the growth rate can be freely selected in a wide range nearly up to the maximum growth rate (.mu..sub.max) if no inhibition occurs. The actual value of .mu..sub.max is highly dependent on the bacterial strain, the expression construct and the growth conditions. The skilled person will understand that the determination of .mu..sub.max is performed under conditions under which the promoter is repressed.

[0108] For a given experimental set up, .mu. can be determined from the growth curve of the culture by plotting biomass concentration (x) as determined by OD.sub.600 or cell wet weight (CWW) against the cultivation time and determining the exponential growth coefficient .mu. based on the equation x=x.sub.0 e.sup..mu.t. The actual value of .mu..sub.max is determined as the growth rate .mu. of an exponentially growing batch culture in the beginning of the batch phase when no substrate limitation occurs, i.e. without supply of additional medium by feeding. The growth rate .mu. can be determined by computing the ratio of the difference between natural logarithm of the total biomass X.sub.2 measured at time t.sub.2 and natural logarithm of the total biomass X.sub.1 measured at time t.sub.1 to the time difference (t.sub.2-t.sub.1): .mu.=(lnX.sub.2-lnX.sub.1)/(t.sub.2-t.sub.1).

[0109] Fed-batch culture allows the maintenance of a constant growth rate (.mu.). In a preferred embodiment the substrate, preferably the major carbon source, is fed during the feed phase according to the exponential increase of the biomass (x). If during the feed phase the substrate is supplied at the same rate it is consumed, the culture is in a quasi steady state, analogous to the cultivation in a continuous culture. Because biomass formation and substrate consumption are correlated over the substrate-referred yield coefficient Y.sub.x/s (biomass [g]/substrate [g]), the substrate quantity (s) per time unit (t) to be supplied is calculated according to the formula ds/dt=.mu./Y.sub.x/x x.sub.0 tote.sup..mu.t, wherein x.sub.0 tot is the total biomass at feed start.

[0110] Therefore, in a preferred embodiment said feeding of said batch culture with said major carbon source is performed with a flow rate, wherein said flow rate increases with an exponential coefficient .mu., and wherein preferably said exponential coefficient .mu. is below .mu.max. Thus, the growth rate of said bacterial host during the feed phase is set to a value which is below .mu..sub.max. In a preferred embodiment said exponential coefficient .mu. is about 30% to 70%, most preferably about 50% of .mu..sub.max. In a specific embodiment of the invention .mu. is set to an absolute value of 0.15 to 0.45 h.sup.-1, more preferably 0.25 to 0.35 h.sup.-1, most preferably .mu. is 0.3 h.sup.-1, provided that the set up of the process is such, that these values are below .mu..sub.max.

[0111] Bacteria are able to utilise a wide range of different substrates. For the purpose of the invention, preferred major carbon sources are glucose and glycerol, preferably glycerol. Although the maximum specific growth rate (.mu..sub.max) of the expression host which can be achieved may be higher with glucose than with glycerol, glycerol causes less acetate formation and provides higher biomass yield per substrate (Y.sub.x/s) and, ultimately, higher yield of the recombinant protein. Furthermore, the handling of the liquid substrate glycerol is easier than that of solid carbon sources like glucose which need to be dissolved in a separate process step.

[0112] As mentioned before, plasmid retention, i.e. the maintenance of the expression plasmid in the bacterial host during the fermentation process, is essential for optimal yield of the recombinant protein. Plasmid retention can be assessed by spreading bacteria cells on a solid medium to form single colonies and testing individual colonies for their antibiotic resistance. For example, a plasmid retention of 100% means that 100 out of 100 tested colonies comprise the specific antibiotic resistance which conferred by the expression plasmid. For the purpose of the invention plasmid retention at the end of the fermentation process is more than 80%, preferably more than 90%, more preferably more than 95%, even more preferably more than 97% and most preferably 100%.

[0113] The optimal growth temperature of a bacterial strain is the temperature at which it reaches its highest maximal growth rate (.mu..sub.max). Under otherwise not limiting conditions for most E. coli strains this temperature is about 37.degree. C. However, growth of the bacterial strain comprising the expression construct at the optimal growth temperature and in the absence of a selective antibiotic may favour the loss of the expression plasmid, whereas plasmid retention is generally improved when the expression strain is grown at lower temperature. Although the maximum growth rate of the expression strain is lower when the strain is grown at temperatures below its optimal growth temperature as compared to growth at the optimal growth temperature, the yield of recombinant protein may be equal or even better at the lower temperature due to improved plasmid retention.

[0114] In one embodiment of the invention, said cultivating of said bacterial host and/or said feeding of said batch culture with said major carbon source and/or said inducing said promoter with an inducer is therefore performed at a temperature below the optimal growth temperature of said bacterial host. In a preferred embodiment said temperature is between 20 and 37.degree. C., preferably between 23 and 35.degree. C., more preferably between 25 and 33.degree. C., even more preferably between 27 and 32.degree. C., still more preferably between 28 and 31.degree. C. Still more preferably said temperature is about 30.degree. C., most preferably said temperature is 30.degree. C.

[0115] The process of the invention comprises a production phase, wherein said production phase is initiated by said inducing said promoter with an inducer. The time point for the initiation of said production phase can be determined based on cultivation time and/or growth parameters.

[0116] The growth of the bacterial host during the fermentation process can be assessed by determining the optical density at 600 nm (OD.sub.600), the cell wet weight (CWW [g/l]) and the cell dry weight (CDW [g/l]). These parameters can be used to define the optimal time point for the start of the production phase by addition of the inducer, preferably lactose, to the medium. It is apparent for the skilled person, that on one hand higher CWW at the beginning of the production phase can be achieved by an extended feed phase and may lead to improved yield of the recombinant protein but that on the other hand over-aged cultures may show insufficient protein expression. The optimal time point for the beginning of the production phase, which is initiated by said inducing of said promoter with an inducer, therefore needs to be determined for the specific production conditions. For example, for expression of Q.beta. CP in E. coli RB791 in a total volume of 2 l, induction is started after ca. 14 h, when OD.sub.600 has reached about 40 to 60. Surprisingly, similar parameters were found for the same process in a 50 l scale, where induction start is also after ca. 14 h when OD.sub.600 has reached about 50.

[0117] Therefore, in one embodiment of the invention, said inducing of said promoter with said inducer is performed 10 h to 16 h after the beginning of said growth phase, preferably after 12 h to 15 h, more preferably after 13 h to 15 h, most preferably after about 14 h, wherein preferably said inducing of said promoter with said inducer is performed when the OD.sub.600 has reached about 40 to 60, preferably about 50.

[0118] In a further embodiment, said inducing of said promoter with said inducer is performed after an extended feed phase, wherein preferably said inducing of said promoter with said inducer is performed 14 h to 20 h after the beginning of said cultivating of said bacterial host in a medium, preferably after 15 h to 18 h, more preferably after 16 h to 17 h, most preferably after about 16.5 h, wherein preferably said inducing of said promoter with said inducer is performed when the OD.sub.600 has reached about 80 to 90, preferably about 85.

[0119] In one embodiment of the invention said inducing of said promoter with said inducer is performed when the OD.sub.600 reached a value of 25 to 60, preferably 25 to 55, more preferably 30 to 50, most preferably 30 to 40. In a specifically preferred embodiment said inducing of said promoter with said inducer is performed when OD.sub.600 is 35.

[0120] In another embodiment of the invention said inducing of said promoter with said inducer is performed after an extended feed phase, when the OD.sub.600 reached a value of 60 to 120, preferably 70 to 110, more preferably 80 to 100, most preferably 80 to 90. In a specifically preferred embodiment the induction is started after and an extended feed phase when OD.sub.600 is about 85, preferably 85.

[0121] Induction with IPTG: In one embodiment of the invention said inducing of said promoter with an inducer is achieved by the addition of IPTG, wherein preferably said feeding of the culture with the major carbon source is continued. Since IPTG is not metabolized by the bacterial host, induction can be achieve by a single addition of IPTG to the desired concentration. Alternatively, induction can be achieved by a continuous flow of IPTG to the culture. In a preferred embodiment induction is performed by addition of IPTG in a single addition or a continuous flow, wherein said feeding of the batch culture with the major carbon source is continued with a constant or an increasing flow rate of said major carbon source exponentially increasing flow rate of the major carbon source.

[0122] Induction with lactose: As described above, the induction of protein expression can alternatively be achieved by the addition of lactose to the culture medium. In one embodiment of the invention, at the beginning of the production phase the exponential feed of the substrate is interrupted and the culture is supplied with a constant flow of induction medium containing 100 to 300 g/l, preferably 100 g/l lactose as the sole carbon source (lactose feed medium). Preferably, the constant flow rate of lactose equals approximately the flow rate of the substrate at the end of the feed phase.

[0123] In a preferred embodiment of the invention said inducing of said promoter with an inducer is achieved by the addition of lactose, wherein preferably said lactose is fed to said batch culture in a continuous flow during and wherein preferably said feeding of said batch culture with said major carbon source is not continued.

[0124] Upon addition of lactose to the culture, the f3-galactosidase activity increases, lactose is converted to allolactose which induces the tac promoter and the expression of the recombinant capsid is initiated. In parallel, allolactose is further metabolised and contributes to the energy supply for the bacterial host. The equilibrium of the feeding rate of the induction medium and the lactose consumption by the cells thus determines the expression rate. The enzymatic reactions involved in this cascade allow to control the process in such a way that the formation of inclusion bodies is minimised. The progress of induction process can be monitored by determining the .beta.-galactosidase activity in the culture, e.g. by a .beta.-Gal Assay Kit (Invitrogen, K1455-01).

[0125] In a more preferred embodiment of the invention said inducing of said promoter with an inducer is achieved by the addition of lactose, wherein preferably said lactose is fed to said batch culture in a continuous flow during and wherein preferably said feeding of said batch culture with said major carbon source is continued.

[0126] Discontinuous addition of inducer: Said inducer can be added to the culture discontinuously by a single addition at the beginning of the production phase or by a few subsequent additions during the production phase. Discontinuous addition of the inducer, especially by a single addition is particularly suited when the inducer is IPTG since IPTG is not metabolized by the bacterial host. Therefore, typically and preferably no replacement of metabolised IPTG is necessary during the production phase. In one embodiment said inducing of said promoter with an inducer is performed by the addition of said inducer, preferably IPTG or lactose, most preferably IPTG, to said medium, wherein said inducer is added to about its final concentration at once by a single addition at the beginning of the production phase, wherein preferably said feeding of said batch culture with said major carbon source is continued. In a preferred embodiment said inducing of said promoter with an inducer is performed by the addition of IPTG to said medium, wherein said IPTG is added to about its final concentration at once by a single addition, wherein preferably said feeding of said batch culture with said major carbon source is continued. Alternatively, said inducing of said promoter with an inducer is performed by the addition of said inducer, preferably IPTG or lactose, most preferably lactose, to said medium, wherein said addition is performed in several steps, preferably in 1 to 5, more preferably in 2 to 4, most preferably in 3 steps during the production phase, wherein preferably said feeding of said batch culture with said major carbon source is continued.

[0127] Continuous addition (feeding) of inducer: Preferably, said inducer is added to the medium in a continuous flow, preferably throughout the production phase. The continuous addition of the inducer is particularly suited for lactose, since lactose is metabolised by the bacterial host and therefore a continuous addition of lactose during the production phase allows to maintain a lactose concentration in the medium which allows for efficient induction of the promoter. In a preferred embodiment, said inducing of said promoter with an inducer is performed by feeding said batch culture with said inducer, wherein preferably said inducer is IPTG or lactose, most preferably lactose, and wherein said feeding is performed in a continuous flow, wherein further preferably said feeding is performed throughout the production phase.

[0128] Co-feeding of inducer and major carbon source: The expression of the recombinant protein is an energy demanding process. To prevent yield loss which might be caused by the excessive consumption of the inducer by the bacterial host and low expression rates resulting thereof, the culture can be additionally supplemented with substrate, preferably the major carbon source, during the production phase, wherein the flow rate of inducer and/or the major carbon source is constant or increasing, preferably constant. When during the production phase the culture is supplemented with substrate at an increasing flow rate, the flow rate is preferably increasing with an exponential rate.

[0129] Co-feeding with constant flow rate: In a preferred embodiment said inducing of said promoter with an inducer is performed by co-feeding said batch culture with said inducer and said major carbon source, wherein said inducer is preferably IPTG or lactose, most preferably lactose, and wherein said major carbon source is glucose or glycerol, preferably glycerol, wherein said inducer, preferably lactose and said major carbon source, preferably glycerol are co-fed to said batch culture at a flow rate, wherein said flow rate is preferably about constant. In a further preferred embodiment said flow rate is chosen to allow feeding of said major carbon source to said batch culture at about the same rate as at the end of the growth phase. In a still further preferred embodiment said inducer, preferably lactose, and said major carbon source, preferably glycerol, are contained in the same medium (co-feed medium). In a further preferred embodiment said co-feed medium is fed to said batch culture with a flow rate, wherein said flow rate is preferably about constant, and wherein further preferably said flow rate is chosen to allow feeding of said major carbon source to said batch culture at about the same rate as at the end of the growth phase. In a very preferred embodiment said inducer is lactose and said major carbon source is glycerol, wherein said lactose and said glycerol are co-fed to said batch culture in a ratio of about 2:1 to 1:4 (w/w).

[0130] In a further preferred embodiment of the invention lactose and said major carbon source, preferably glycerol, are co-fed to said batch culture in a ration of 0:1 to 1:0 (w/w), preferably about 2:1 to about 1:4 (w/w), more preferably about 1:1 to 1:3 (w/w), most preferably the ratio is about 1:3 (w/w). In a preferred embodiment the ratio of lactose and the major carbon source, preferably glycerol, is 1:1 (w/w). In another preferred embodiment the ratio of lactose and the major carbon source, preferably glycerol, is 1:3 (w/w). In a more preferred embodiment said co-feed medium comprises ca. 200 g/l lactose and ca. 200 g/l glycerol. In a still more preferred embodiment the co-feed medium comprises ca. 100 g/l lactose and ca. 300 g/l glycerol.

[0131] Co-feeding with increasing flow rate: Alternatively, said inducing of said promoter with an inducer is performed by co-feeding said batch culture with said inducer and said major carbon source, wherein said inducer is preferably IPTG or lactose, most preferably lactose, and wherein said major carbon source is glucose or glycerol, preferably glycerol, wherein said inducer, preferably lactose and said major carbon source, preferably glycerol are co-fed to said batch culture at a flow rate, wherein said flow rate is increasing, wherein said flow rate may increase with a linear or with an exponential characteristic, wherein preferably the initial flow rate is chosen to to allow feeding of said major carbon source to said batch culture at about the same rate as at the end of the growth phase.

[0132] Further alternatively said inducing of said promoter with an inducer is performed by co-feeding said batch culture with said inducer and said major carbon source, wherein said inducer is preferably IPTG or lactose, most preferably lactose, and wherein said major carbon source is glucose or glycerol, preferably glycerol, wherein said inducer, preferably lactose is fed to said batch culture at a first flow rate, and wherein said major carbon source, preferably glycerol is fed to said batch culture at a second flow rate, wherein said first flow rate is constant or increasing, preferably constant, and wherein said second flow rate is constant or increasing, preferably increasing, wherein preferably the initial value of said second flow rate is chosen to to allow feeding of said major carbon source to said batch culture at about the same rate as at the end of the growth phase. In a very preferred embodiment said inducer is lactose and said major carbon source is glycerol, wherein said lactose and said glycerol are co-fed to said batch culture in a ratio of about 2:1 to 1:4 (w/w).

[0133] The growth of the bacterial host as determined by CDW, CWW or OD.sub.600 continues during the production phase at a growth rate which is lower than that during the growth phase and which is decreasing with the process time. In a further embodiment of the invention, said inducing said promoter with an inducer is performed by co-feeding said inducer, preferably lactose and said major carbon source, preferably glycerol, to said batch culture with an increasing flow rate, preferably with a flow rate wherein the incremental increase of the flow rate is adapted to the actual growth rate of the culture. In a further preferred embodiment said inducer, preferably lactose, and said major carbon source, preferably glycerol, are contained in the same medium (co-feed medium), wherein preferably the ratio between lactose and glycerol in said medium (co-feed medium) ranges from about 0:1 to 1:0 (w/w), preferably about 2:1 to about 1:4 (w/w), more preferably about 1:1 to 1:3 (w/w), most preferably the ratio is about 1:3 (w/w). In a preferred embodiment the ratio of lactose and the major carbon source, preferably glycerol, is 1:1 (w/w). In another preferred embodiment the ratio of lactose and the major carbon source, preferably glycerol, is 1:3 (w/w). In a more preferred embodiment said medium (co-feed medium) comprises ca. 200 g/l lactose and ca. 200 g/l glycerol. In a still more preferred embodiment the induction medium comprises ca. 100 g/l lactose and ca. 300 g/l glycerol.

[0134] In one embodiment of the invention said inducing of said promoter with an inducer is performed by co-feeding said inducer, preferably lactose and said major carbon source, preferably glycerol to said batch culture, wherein said inducer, preferably lactose and said major carbon source, preferably glycerol are contained in separate media which are separately fed to said culture.

[0135] At the end of the production phase the cells are harvested by centrifugation. Typically, cells are harvested about 5 h after induction start, when a final OD.sub.600 of 90 to 130 is reached. Further extension of the production phase leads to higher OD.sub.600 and CWW values and therefore to further improved yield of the expression construct.

[0136] Harvested cells may be suspended in a storage buffer and stored at -80.degree. C. for further processing.

[0137] The total protein content of the cells is determined after cell lysis by SDS PAGE or LDS PAGE and comparison with a protein standard. The content of soluble protein is determined by HPLC. The identity of the expressed capsid protein is determined by western blotting. The concentration of assembled VLPs can be analysed by size exclusion chromatography (Example 18). VLP can preparatively be purified from lysed cells by chromatographic methods.

[0138] Scale-up of the process of the invention to large volumes is possible with only minor adaptations. The invention encompasses culture volumes in the range of 100 ml up to 6000 l. Preferred culture volumes are 40 to 100 l, most preferably about 50 l. It is apparent for the skilled person that larger culture volumes in particular require larger volumes of the preculture which is used for inoculation. For example, a preculture may be performed in two ore more steps with increasing preculture volume. To ensure plasmid retention in large culture volumes, the precultures which are used as inoculum may contain an antibiotic to maintain selection pressure. The skilled person is aware that plasmid retention can further be improved by reducing the number of generations which is necessary to reach the desired final cell density. Therefore, it is advantageous to inoculate the precultures and the batch cultures with high cell densities. In a preferred embodiment the initial OD.sub.600 of the preculture is 0.1 to 0.4, preferably about 0.3.

[0139] In one embodiment, prior to said cultivation step, said process further comprises the step of introducing said bacterial host into a medium, wherein said introducing is performed with an inoculum, wherein said inoculum is produced in a preculture process comprising the step of growing said bacterial host in a medium comprising an antibiotic, preferably kanamycin. More preferably, said preculture process comprises the steps of growing said bacterial host in a first medium comprising an antibiotic, preferably kanamycin, and diluting said first medium comprising the bacterial host with a second medium to an OD.sub.600 of 0.1 to 0.4, preferably about 0.3, wherein said second medium is essentially free of an antibiotic, and further cultivating said bacterial host.

[0140] Furthermore, it is apparent for the skilled person, that the fermentation process of the invention is an aerobic process which requires adequate oxygen supply of the bacteria in the culture. The oxygen demand of the bacterial host is, inter alia, increasing with increasing cell density and increasing growth rate. Depending on the total volume and the oxygen demand of the bacterial host, oxygen can, for example, be supplied by stirring and/or by aeration with air. Alternatively, oxygen can also be supplied by aeration with pure oxygen or a mixture of pure oxygen with any other gas, preferably air, wherein pure oxygen refers to the technically pure gas as commonly available for technical purposes. A further possibility of supplying oxygen to the bacterial host is increasing the oxygen partial pressure in the medium by increasing the pressure in the fermenter.

[0141] In a preferred embodiment of the invention, said cultivating said bacterial host and/or said feeding of said batch culture and/or said inducing of said promoter with an inducer is performed under conditions, wherein said bacterial host is supplied with oxygen, preferably by aeration with air, most preferably by aeration with air in a constant flow, wherein preferably said oxygen is supplied throughout the entire process, most preferably throughout the lag-, growth- and production phase, and wherein further preferably the partial pressure of oxygen is monitored in the culture medium and wherein the bacterial host is alternatively or additionally supplied with oxygen by aeration with pure oxygen, preferably when the partial pressure of oxygen in the medium (pO.sub.2) is below a certain threshold. In a specifically preferred embodiment said threshold of pO.sub.2 is in the range of 0% to 60%, preferably 10% to 50%, more preferably 20% to 45% most preferably said threshold is about 40%.

[0142] Oxygen supply, preferably by aeration with air and/or pure oxygen to maintain the preferred pO.sub.2 as described above, is routinely applied in the process of the invention, preferably for culture volumes of 2 l and more. Aeration with oxygen in the described manner is especially preferred in the scaled-up process, most preferably at 40 to 100 l and above. Therefore, one embodiment of the invention is a process for expression of a recombinant capsid protein of a bacteriophage or a mutant or fragment thereof being capable of forming a VLP by self-assembly, said process comprising the steps of: a) introducing an expression plasmid into a bacterial host, wherein said expression plasmid comprises an expression construct, wherein said expression construct comprises (i) a first nucleotide sequence encoding said recombinant capsid protein, or mutant or fragment thereof, and (ii) a promoter being inducible by lactose; b.) cultivating said bacterial host in a medium comprising a major carbon source; wherein said cultivating is performed in batch culture and under conditions under which said promoter is repressed by lad, wherein said lad is overexpressed by said bacterial host; c.) feeding said batch culture with said major carbon source; and d.) inducing said promoter with an inducer, wherein said feeding of said batch culture with said major carbon source is continued;

[0143] wherein throughout steps b.) to d.) of said process oxygen is supplied to said bacterial host by a pO.sub.2 in said medium of at least about 10% to 50%, preferably about 40%, and wherein further preferably said oxygen is supplied by aeration with air, pure oxygen, or a mixture of both, preferably by a mixture of air and pure oxygen.

EXAMPLES

Example 1

Cloning Strategy for the Expression Plasmid pTac-nSD-Qb-mut (SEQ ID NO:1)

[0144] The coat protein-encoding gene (C) of E. coli RNA bacteriophage QB is amplified from plasmid pSDQb-mut (SEQ ID NO:33). The plasmid contains the sequence of gene C coding for the 133-aa Q.beta. coat protein (CP) and the 329-aa read through protein (A1). To prevent read-through, nucleotides 445-450 according to NCBI GenBank Acc. No. M99030 TGAACA (SEQ ID NO:31) are replaced by the sequence TAATGA (SEQ ID NO:32).

[0145] The coat protein-encoding gene C from plasmid pSDQb-mut is amplified by PCR. Oligonucleotide Qb-FOR3/2 (SEQ ID NO:34) with an internal EcoRI site and a synthetic Shine-Dalgarno (SD, SEQ ID No:4) sequence anneals to the 5' end of the QB CP gene. Oligonucleotide Qblang-REV2/2 (SEQ ID NO:35) contains an internal HindIII site and primes to the 3' end of the noncoding region of gene C. The 1054 bp amplified PCR fragment includes nucleotides 46-1062 of NCBI GenBank Acc. No. M99039 (except the nucleotide changes described above) and the synthetic SD sequence. The PCR fragment is digested with the restriction enzymes HindIIII/EcoRI and the resulting 1036 bp fragment is inserted into the HindIII/EcoRI restriction sites of a modified pKK223-3 vector (Pharmacia, NCBI GenBank Acc. No.: M77749, SEQ ID NO:27). In this modified pKK223-3 vector the ampicillin resistance gene is replaced with the kanamycin resistance gene of vector pUC4K (Pharmacia, NCBI GenBank Acc. No.: X06404, SEQ ID NO:37).

[0146] Vector pTac-nSDQb-mut (SEQ ID NO:33) differs from vector pTacQb-mut in the Shine-Dalgarno sequence. This Shine-Dalgarno sequence (nSD, SEQ ID NO:3) is introduced by amplifying the Q.beta. coat protein-encoding gene C via PCR from plasmid pTacQb-mut. Oligonucleotide nSDQb-mutEcoRlfor (SEQ ID NO:36) with an internal EcoRI site and the corresponding synthetic Shine-Dalgarno (nSD) sequence anneals to the 5' end of the Q.beta. CP gene. Oligonucleotide Qblang-REV2/2 (SEQ ID NO:35) contains an internal HindIII site and primes to the 3' end of the noncoding region of gene C. The 1054 bp amplified PCR fragment includes nucleotides 46-1062 of NCBI GenBank Acc. No. M99039 (except the nucleotide changes described above) and the synthetic nSD sequence. The PCR fragment is digested with the restriction enzymes HindIII/EcoRI and the resulting 1036 bp fragment is inserted into the HindIII/EcoRI restriction sites of a modified pKK223-3 vector (Pharmacia, NCBI GenBank Acc. No.: M77749, SEQ ID NO:27). In this modified pKK223-3 vector the ampicillin resistance gene is replaced with the kanamycin resistance gene of vector pUC4K (Pharmacia, NCBI GenBank Acc. No.: X06404, SEQ ID NO:37).

Example 2

Cloning Strategy for the Expression Plasmid pTac-nSD-AP205 (SEQ ID NO:30)

[0147] The coat protein-encoding gene of Acinetobacter bacteriophage AP205 is amplified from plasmid pAP205-58. This plasmid contains the sequence of the coat protein gene (corresponding to nucleotides 1908-2303 of NCBI GenBank Acc. No. AF334111) coding for the 131-amino acid capsid protein of bacteriophage AP205.

[0148] The coat protein-encoding gene is amplified by PCR. Oligonucleotide nSDAP238-EcoRIfor (SEQ ID NO:38) with an internal EcoRI site and a synthetic Shine-Dalgarno (nSD) sequence anneals to the 5' end of the coat protein gene. Oligonucleotide AP238HindIIIrev (SEQ ID NO:39) contains an internal HindIII site and primes to the 3' end of the coat protein gene. This oligonucleotid introduces a second stop codon behind the naturally occurring stop codon of the coat protein. The 438 bp amplified PCR fragment includes nucleotides 1908-2303 of NCBI GenBank Acc. No. AF334111 and the synthetic nSD sequence. The PCR fragment is digested with the restriction enzymes HindIII/EcoRI and the resulting 420 bp fragment is inserted into the HindIII/EcoRI restriction sites of a modified pKK223-3 vector (Pharmacia, NCBI GenBank Acc. No.: M77749, SEQ ID NO:27). In this modified pKK223-3 vector the ampicillin resistance gene is replaced with the kanamycin resistance gene of vector pUC4K (Pharmacia, NCBI GenBank Acc. No.: X06404, SEQ ID NO:37).

Example 3

Expression of Q.beta. CP Under Control of the tac Promoter and nSD

[0149] The E. coli strain RB791 was transformed with plasmids pTac-nSD-Qb-mut (SEQ ID NO:1). The clone was grown in shake flasks. Each flask contained 100 ml of R40 medium (main culture medium, Hypep 7455, glycerol, see Example 5) with kanamycin (25 .mu.g/ml) and was inoculated with over night cultures at a start OD.sub.600 of 0.3. The shake flasks were incubated for 4 h (OD.sub.600 between 4 and 5) at 30.degree. C. and an agitation of 220 rpm. The induction was carried out with 0.5% of lactose for 4 h. Protein production was determined by SDS-PAGE. The gel showed a strong protein band which was identified as Q.beta. CP.

Example 4

Expression of AP205 CP Under Control of the tac Promoter and SD vs. nSD

[0150] 9 clones of pTac-nSDAP205 (SEQ ID NO:30) and 6 clones of pTac-SDAP205 were screened in shake flasks. pTac-SDAP205 (SEQ ID NO:40) is identical to pTac-nSDAP205 but comprises the Shine-Dalgarno sequence of SEQ ID NO:4 instead of that of SEQ ID NO:3. Each flask contained 50 ml of R40 medium (main culture medium, Hypep 7455, glycerol, see Example 5) with kanamycin (25 .mu.g/ml) and was inoculated with over night cultures at a start OD.sub.600 of 0.3 (for pTac-nSDAP205) or 0.4 (pTac-SDAP205). The shake flasks were incubated for 4 h at 30.degree. C. and an agitation of 220 rpm. The induction was carried out with 0.5% of lactose. Protein production was determined by SDS-PAGE. For all tested clones expression of AP205 CP was significantly stronger from pTac-nSDAP205 than from pTac-SDAP205.

Example 5

Composition of Culture Media

Culture media were composed as described in Table 1.

TABLE-US-00001

[0151] TABLE 1 Composition of Culture media. Concentrations in [g/L] Main Feed Induction Main Medium + Medium + Medium + 20% Main Medium + Hypep + 50% Glycerol + 20% Medium + Bacto Hypep Glycerol Glycerol Lactose YE + Glycerol R27 R40 R41 R42 R43 Component Na.sub.2HPO.sub.4 2H.sub.2O 2.5 2.5 2.5 2.5 2.5 KH.sub.2PO.sub.4 3 3 3 3 3 K.sub.2HPO.sub.4 5.2 5.2 5.2 5.2 5.2 Citrate 3.86 3.86 3.86 3.86 3.86 (NH.sub.4)2SO.sub.4 4 4 4 4 4 Vit B1 0.01 0.01 0.02 0.02 0.01 CaCl.sub.2 2H.sub.2O 0.0147 0.0147 0.0147 0.0147 0.0147 MgSO.sub.4 7H.sub.2O 0.5 0.5 9 9 0.5 FeCl.sub.3 6H.sub.2O 0.054 0.054 0.054 0.054 0.054 CoCl.sub.2 6H.sub.2O 0.0005 0.0005 0.0005 0.0005 0.0005 MnCl.sub.2 4H.sub.2O 0.003 0.003 0.003 0.003 0.003 CuCl.sub.2 2H.sub.2O 0.0003 0.0003 0.0003 0.0003 0.0003 H.sub.3BO.sub.3 0.003 0.003 0.003 0.003 0.003 Na.sub.2MoO.sub.4 2H.sub.2O 0.0005 0.0005 0.0005 0.0005 0.0005 Zn(CH.sub.3COO).sub.2 2H.sub.2O 0.0026 0.0026 0.0026 0.0026 0.0026 Glucose 5 -- -- -- -- Glycerol -- 5 500 200 5 Lactose anhydrous -- -- -- 200 -- HyPep 7455 5 5 -- -- -- Bacto Yeast Extract -- -- -- -- 5

Example 6

Expression of Q.beta. CP in a Fed-batch Process (2 L Scale)

[0152] The fermentation process was performed in a bioreactor (Applikon 5 L dished bottom) equipped with 2 disc stirrer (O6 cm), baffles (3.times.16 cm), pH-, pO2-, and temperature control, and fermenter software BioXpert Version 2.22

[0153] 5 .mu.L cryo culture of RB791 transformed with plasmids pTac-nSD-Qb-mut were inoculated in 100 mL Erlenmeyer flasks containing 50 mL medium R40 (25 .mu.g/mL kanamycin) and cultivated for 14 h at 30.degree. C. and 220 RPM over night. After 14 h an OD.sub.600 value of 6.0 was reached. For batch fermentation, 2 L of medium (R40) were pumped into the bioreactor. In Table 2 the cultivation parameters are listed.

TABLE-US-00002 TABLE 2 Parameter set points for batch phase. Parameter Set point Unit Stirrer speed 1000 [rpm] Air supply 2.5 [L/min] O2-supply, maximal 2 [L/min] Temperature 30 [.degree. C.] O2-saturation >40 [%] pH 6.8 [--]

[0154] The bioreactor was inoculated with 100 mL inoculum. Samples of 2 mL were taken, OD.sub.600 determined and centrifuged at 14'000 RPM. Pellet and supernatant were separated and frozen for further analysis. The biomass concentration [g/L] was calculated using the following equation:

OD.sub.600.times.0.45 [g.times.L.sup.-1.times.OD.sub.600.sup.-1]=biomass [g/L].

[0155] The Qbeta content in percent of the total protein content was calculated as follows, assuming, that 50% of the E. coli biomass is protein:

Biomass [g.times.L.sup.-1]/2=total protein [g.times.L.sup.-1]

Qbeta [g.times.L.sup.-1]/total protein [g.times.L.sup.-1].times.100=Qbeta/total protein [%].

[0156] In the fed-batch mode, which followed the batch mode, a feeding phase was added. In the feeding phase substrate is supplied to the cells in the reactor according to a defined profile. The feed profile depends on the selected growth rate .mu.., the yield coefficient biomass to glycerol (Y.sub.x/glycerol), the volume (Vf), and the concentration of substrate in the feed (cf). substrate concentration. The feed was calculated using the following equation:

[0157] Feed equation

mf=(.mu./Y.sub.x/s+m) Vf.times.Xf.times.e.sup..mu.t

pump=(mf/cf+b)/a

[0158] mf=mass flow [g/h]

[0159] .mu.=specific growth rate [l/h]

[0160] Y.sub.x/Glycerol=Yield biomass to glycerol [g/g]

[0161] m=maintenance energy [gg.sup.-1h.sup.-1]

[0162] Vf=Volume at feed start

[0163] Xf=Biomass at feed start

[0164] cf=Concentration of substrate in feed [g/mL]

[0165] a+b=offset/slope of pump calibration equation

[0166] For the determination of the calibration parameters a and b, a pump calibration was carried out. In addition, the feed tube with feed bottle was clamped into the feed pump and the pump was run with 7, 14 and 21% pump performance. The pumped feed volume per time was noted. In a resulting diagram of the relation of pump performance [%] to pumped feed solution [mL/h], the slope (a) and the Y-axis section (b) was determined. On the bioreactor the parameters in Table 3 were set for fed-batch cultivation.

TABLE-US-00003 TABLE 3 Parameters for fed-batch cultivation in bioreactor. Parameter Set point Unit Stirrer speed 1000 [rpm] Air supply 2.5 [L/min] O.sub.2-supply, maximal 2 [L/min] Temperature 30 [.degree. C.] O.sub.2-saturation >40 [%] pH 6.8 [--]

[0167] After reaching a process time of approximately 7 h (end of batch) the feed pump was turned on automatically. After further 7 h cultivation, when the M.sub.oo reached 55-60, the feed medium (for biomass propagation) was exchanged with the induction medium R42 (for biomass propagation and induction). After 5 h feeding of R42 was stopped and the culture was harvested by centrifugation.

Analysis of Process Parameters

[0168] The following process parameters were routinely analysed. The pO.sub.2, pH, temperature and stirrer speed were measured online throughout the process time. The optical density was measured offline at 600 nm. The determination of the .beta.-galactosidase activity was performed using a .beta.-Gal Assay Kit (Invitrogen, cat. no. K1455-01). The activity was specified as units per mL OD.sub.600=1.0. It is defined as the quantity of Ortho-Nitrophenyl-.beta.-D-Galactopyranosid (ONPG) in nmol, which is hydrolysed per minute and mL bacteria suspension (OD.sub.600=1.0). The accumulated product was analysed by SDS-PAGE, the total protein content (soluble and insoluble protein) was determined and using HPLC analysis, the soluble fraction was measured. Cell disruption of E. coli was performed in lysis buffer (50 mM glucose, 25 mM tris/HCl (pH 8), 15 mM EDTA (pH 8.0)) with and ultrasonic homogeniser (Bandlin Sonoplus, HD2070). 250 .mu.L bacteria suspension with an OD.sub.600 of 50 were centrifuged with 14000 RPM for 10 min. The pellet was resuspended in 250 .mu.L lysis buffer (vortex) and placed at room temperature for 5 min Afterwards, the cells were disrupted for 20 s with ultrasonic at 10% device performance (cells on ice) and then the cell suspension was centrifuged at 14000 RPM, 10 min The supernatant (soluble protein) was then analysed by SDS-PAGE and HPLC.

[0169] Samples before induction and at end of production (after 5h induction) were taken from the bioreactor for analysis of Q.beta. formation analyzed by SDS-PAGE standardized to OD 5.0. At the end of cultivation, 1.9 l of the culture was harvested. After centrifugation, the following cell pellets were obtained in three independent reactor runs: 1.) End OD.sub.600 of 84: 194 g CWW; 2.) End OD.sub.600 of 88: 200 g CWW; 3.) End OD.sub.600 of 86: 201 g CWW.

[0170] The plasmid retention in run 1 and 2 was 100% at induction start and 100% at harvest. Based on comparison with a Q.beta. CP standard on SDS-PAGE the yield was roughly estimated to be about 5 g/l Q.beta. CP. HPLC analysis revealed a concentration of about 6 g/l Q.beta. VLP.

Example 7

Selection of Carbon Source and Bacterial Strain

[0171] Glucose and glycerol as carbon sources were compared. In order to test the growth behaviour of each of the strains DH20 and RB791 on these carbon sources, shake flask experiments were conducted with medium containing glucose (R27) and medium containing glycerol (R40). Both media were supplemented with 25 .mu.g/ml kanamycin. Each culture was started with an initial OD.sub.600 of 0.3. Induction was performed by adding 0.5% lactose. The maximum specific growth rates (.mu..sub.max) and the yield coefficients (Y.sub.x/s) were determined and are listed in Table 4. RB791 grew faster on both, glucose and glycerol. In addition, the resulting yield coefficients were higher. Although glucose allowed higher maximum specific growth rates (.mu..sub.max) the yield coefficients (Y.sub.x/s) was higher for glycerol.

TABLE-US-00004 TABLE 4 Maximum specific growth rates and the yield coefficients of the cultivation experiments with RB791 and DH20 on glucose and glycerol. Yield Max. coefficient spec. (Y.sub.x/s) growth biomass Value after 4.5 h Culture Time rate from Carbon Acetate (.mu..sub.max) substrate Strain source OD.sub.600 [g1.sup.-1] [h.sup.-1] [g/g] RB791 glucose 6.24 0.44 0.71 0.72 glycerol 4.04 0.21 0.62 0.86 DH20 glucose 2.52 0.42 0.51 0.71 glycerol 2.82 0.25 0.50 0.81

Example 8

Determination of Optimal Temperature

[0172] The influence of temperature on product formation was investigated. Two shake flask cultures were inoculated and incubated at 30.degree. C. and 220 rpm. After an OD.sub.600 of 5 was reached, the cultures were induced with lactose. Subsequently, one culture was continued to be incubated at 37.degree. C. and the other culture at 23.degree. C. Results of the SDS-PAGE revealed that expression levels at 4 and 5 h after induction are higher in the culture induced at 37.degree. C. Induction of the cultures for 19 h showed a higher Q.beta. level in the cultures induced at 23.degree. C.

Example 9

Induction by Co-Feed of Lactose and Glycerol

[0173] A feed solution of 20% glycerol and 20% lactose was composed (R42) and applied to fermentation as described in Example 6 at induction start. FIG. 1 provides an overview over relevant process parameters throughout the entire process time. Expression was induced at 13.5 h at an OD.sub.600 of about 55. Upon induction, the feed pump rate was set to constant. Glycerol did not accumulate with feeding. Lactose accumulated to 4 g/l and then it started to diminish. The .beta.-galactosidase activity rose to 10 U/ml and decreased thereafter. Compared with the previous fermentation runs a.) lactose applied as a single lactose pulse at induction start, no feeding; b.) continuous lactose feed without glycerol, the activity was with 7 U/ml higher and the maximum activity was already reached after 2 h as compared to 4 h in runs a.) and b.).

Example 10

Plasmid Retention

[0174] The effect of the following operating conditions on the plasmid retention was tested in the process described in Example 6: 1.) Preculture starting volume, 2.) Kanamycin in the preculture, 3.) Growth and/or induction at 37.degree. C. vs. 30.degree. C. The results are summarised in Table 5. Precultures were started with volumes of 5 .mu.l out of the cell bank vial. Inoculation of a small volume allowed growth of a preculture over-night. The preculture for QT0103_F8 contained 25 mg/l kanamycin, whereas the preculture for QT0103_F7 did not contain any kanamycin. Both fermentations were operated at 30.degree. C. and induced for 5 h. Judging from the plasmid retentions before and after 5 h induction, supplementing the preculture with kanamycin has a positive effect on plasmid retention. Plasmid retention remained at 98% before and after 5 h induction. In contrast, plasmid retentions reached only values of 80% when kanamycin was omitted from the preculture. For a subsequent run, QT0203_F7, the preculture was also started with 5 .mu.l and grown in kanamycin containing medium. The resulting fermentation in the bioreactor was operated at 37.degree. C. from the beginning. Operation at 37.degree. C. had a detrimental effect on the plasmid stability. While the plasmid retention was at 99% before induction, it dropped to 0% after 5 h induction. In order to test whether a shorter preculture and thus, less generations, would improve the plasmid retention after 5 h induction, a set of precultures were started with 300 .mu.l volume from a thawed cell bank vial and grown in kanamycin free medium. Two fermenters were operated at 30.degree. C. for the whole run. An additional two fermenters were operated first at 30.degree. C. for cell growth and than switched to 37.degree. C. for the production phase. The resulting plasmid stabilities were all at 100% before and 5 h after induction.

TABLE-US-00005 TABLE 5 Summary of plasmid retention before and 5 h after induction obtained under different operating conditions in terms of generations in the preculture, with and without kanamycin in the preculture, and growth and/or induction at 37.degree. C. Plasmid Plasmid Preculture Kana- retention retention Starting mycin in before after 5 h Bioreactor Culture pre- induc. induc. run Volume culture [%] [h] Remarks QT0103_F8 5 25 mg/L 98 98 whole process at 30.degree. C. QT0103_F7 5 no 80 80 whole process at 30.degree. C. QT0203_F7 5 25 mg/L 99 0 Bioreactor run at 37.degree. C. QT0603_F7 300 no 100 100 whole process at 30.degree. C. QT0703_F8 300 no 100 100 Induction at 37.degree. C., rest of the process at 30.degree. C. QT0803_F9 300 no 100 100 whole process at 30.degree. C. QT0903_F10 300 no 100 100 Induction at 37.degree. C., rest of the process at 30.degree. C.

Example 11

Variation in Time Point of Induction

[0175] In a process essentially as described in Example 6 the exponential feed profile was programmed to start 7 h after the inoculation of the bioreactor. Under standard conditions, the scheduled time for induction was at 14 h process time. In order to test the effect of variations in the time point of induction on the final cell densities, one culture was induced at 13.5 h (resulting in 6.5 h of exponential feed) and another culture at 14.5 h (resulting in 7.5 h of exponential feed). One culture induced at the regular 14 h time point served as a control (7 h of exponential feed). Results are summarised in Table 6. Cell density increased with increasing length of feeding.

[0176] Judged from a linear regression analysis of the available data points for final CWW, a linear relationship appears to exist (r.sup.2=0.92).

TABLE-US-00006 TABLE 6 Variations in time point of induction: effect on final cell density in terms of OD.sub.600 and CWW. Process Time Duration of Final Point of Exp. Feed Final CWW Reactor Induction Phase [h] OD.sub.600 [g/L] F2 13 h 32 min 6.5 83.4 116.5 Fl 14 h 02 min 7.0 82.4 122.5 F3 14 h 29 min 7.5 100.4 141.1

Example 12

Variation in Time Point of Harvest

[0177] Harvest of the culture in a process essentially as described in Example 6 is performed manually. Under standard conditions, the scheduled time for harvest was at 19 h process time. The operation "Harvest" involves the manual ending of the bioreactor operations. In order to test the effect of variations in the time point of harvest on the final cell densities, one culture was harvested at 18.8 h (resulting in 4.8 h of induction) and another culture at 19.5 h (resulting in 5.5 h of induction). One culture harvested at the regular 19 h time point served as a control (5 h of induction). Results are summarized in Table 7. Cell density increased with increasing length of induction because the cells are still growing while induced.

TABLE-US-00007 TABLE 7 Variation in time point of harvest: effect on final cell density in terms of OD600 and CWW. Process Time Length of Final Point of Induction Final CWW Reactor Harvest [h] OD.sub.600 [g/L] F5 18 h 50 min 4.8 91.4 122.4 F4 19 h 00 min 5.0 92.2 127.5 F6 19 h 30 min 5.5 96.0 132.4

Example 13

Effect of Temperature

[0178] The effect of fermentation temperature in a process essentially as described in Example 6 was investigated by running 6 fermentations at 5 different temperature setpoints. Results are summarized in Table 8. Final cell densities were sensitive to the fermentation temperature with an optimum at a temperature of 30.degree. C.

TABLE-US-00008 TABLE 8 Summarized results of different temperature setpoints on final cell density in terms of 0D600 and CWW. Temperature Final CWW Reactor [.degree. C.] Final OD.sub.600 [g/L] F5 25.0 37.8 62 F4 27.5 80.0 117 F3 30.0 92.8 123 F4 30.0 92.4 125 F5 32.5 85.0 111 F6 35.0 79.6 107

Example 14

Scaled-Up Fermentation (50 1)

[0179] The process described in Example 6 was scaled up to a volume of 50 l order to evaluate scale-up capability from the 2 L working volume bioreactor system to a larger volume. Key process parameters for the scaled-up process are summarized in Table 9.

TABLE-US-00009 TABLE 9 Process parameters of in 50 L bioreactor. Time Culture Step Description [h] OD.sub.600 Preculture 1 300 .mu.l from cell bank vial are transferred -11* 5.0 into 100 ml preculture medium contained in 500 mL shake flask and cultured for 16 h Preculture 2 Calculate the required volume for -5* 4.0 transfer in order to start with initial OD.sub.600 of 0.3 in 750 ml. Tranfer calculated volume (e.g. 50 ml) into 750 ml preculture medium contained in 5000 mL shake flask Inoculation Pooled calculated volume (e.g. 1.4 L) is 0 of Bioreactor transferred into the 50 L Bioreactor. Initial volume: =40 L Induction The exponential feeding profile is 14 46 Start switched to constant and feed is switched to induction feed End of Culture is completed after 5 h 19 128 Culture of induction *Relative to the time of bioreactor inoculation.

[0180] It was necessary to have two preculture expansion steps. In the first step, the cells were expanded as established for the 2 L process (Example 6). After this step, cells were split into two 5000 ml shake flask cultures, containing 750 ml medium each. Further expansion was performed for 5 h. The cultures in the 50 L bioreactors were performed with the same time profile as in the 2 L system (Example 6). OD.sub.600 at induction start was 46, the final OD.sub.600 was 128. Plasmid retention was 100% before induction and 98% at the end of culture. The concentration of Q.beta. CP protein in the medium at the end of culture was roughly estimated 8 g/l using SDS-PAGE. The total amount of Q.beta. was estimated about 300 g for this reactor run.

Example 15

Effect of Extended Exponential Feed

[0181] The exponential feeding phase for fermentations performed according to Examples 6 or 14 was 7 h. After this time the cells reached a density for induction, which increased during induction to the targeted maximum 0D.sub.600 of around 100 to 130 as final cell density. Final OD.sub.600, final CWW, final CDW, plasmid retention at induction start and harvest and Q.beta. concentration at the end of culture are determined for reactor runs performed as described in Examples 6 and 14, preferably as in Example 14, wherein the exponential feeding phase is extended to a duration up to 11 h, preferably to 10 h.

Example 16

Effect of Increased Feeding During Production

[0182] Example 9 demonstrates that the glycerol does not accumulate during production phase, indicating that production might be limited by the feeding rate of induction medium. Effect of extended feeding rate of induction medium on final OD.sub.600, final CWW, final CDW, plasmid retention at induction start and harvest and Q.beta. concentration at the end of culture is determined in reactor runs as described in Example 6 and 14, preferably as in Example 14, wherein the feeding rate during production is increased. Alternatively or additionally, the ratio between lactose and glycerol in the feed medium shifted towards a higher glycerol and a lower lactose concentration.

Example 17

HPLC Analysis of Q.beta. CP

[0183] Q.beta. CP was measured with an HPLC system as follows: A sample containing Q.beta. CP was diluted appropriately in 1.times. reaction buffer (50 mM tris(hydroxymethyl)aminomethane buffer pH 8.0) containing 10 mM 1,4-Dithio-DL-threitol and incubated for 15 min at 50.degree. C. in a thermomixer. After incubation the sample was centrifuged and the supernatant was stored at 2.degree. C. to 10.degree. C. until HPLC analysis. 10 to 100 .mu.l of the sample were injected.

[0184] Q.beta. was quantified with a regression curve of known Q.beta. standards regressed to the HPLC peak area detected at 215 nm after elution from a C.sub.4 reversed phase column, 300 .ANG., 5 .mu.m, 4.6.times.150 mm, Vydac Inc., Hesperia, USA (Cat. No. 214TP5415) thermally equilibrated at 50.degree. C. The flow rate through the system was 1 ml/min consisting of mobile phase A (0.12% trifluoroacetic acid in water) and mobile phase B (0.12% trifluoroacetic acid in acetonitrile) with the following gradient of phase B: 0 to 2 min constant at 40%, 2 to 8 min linear increase to 50%, 8 to 10 min constant at 50%, 10 to 10.1 min linear decrease to 40%, and 10.1 to 12 min constant at 40%.

Example 18

Determination of Q.beta. VLP by Analytical Size Exclusion Chromatography

[0185] Analysis of Q.beta. particles by analytical size exclusion chromatography was performed using a Tskge1G5000 PW.sub.XL-column (10 .mu.m, 7.8.times.300 mm, TosoH Biosep; Cat.-No. 08023) equilibrated in phosphate buffered saline (20 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4, 150 mM NaCl pH 7.2). Elution was performed by an isocratic gradient for 20 min at 0.8 ml/min in phosphate buffered saline. The Qbeta concentration was determined from a regression curve of known Q.beta. standards regressed to the HPLC peak area detected at 260 nm.

Example 19

Effect of Extended Exponential Feed

[0186] The exponential feeding phase for fermentations performed according to Examples 6 or 14 was 7 h. After this time the cells reached a density for induction, which increased during induction to the targeted maximum OD.sub.600 of around 100 to 130 as final cell density. Final OD.sub.600, final CWW, plasmid retention before induction and at harvest and Q.beta. concentration at the end of culture were determined for reactor runs performed as described in Examples 6 and 14, preferably as in Example 14, wherein the exponential feeding phase was extended to a duration up to 12 h. In addition, the concentration of glycerol and lactose in the induction feed were changed to 300 g/L and 100 g/L respectively. The results are summarized in Table 10.

TABLE-US-00010 TABLE 10 OD.sub.600 and CWW at the end of cultivations, Plasmid Retention before induction and at the end of cultivations as well as the peak oxygen mass flow. The cultivations were conducted with different duration of exponential feeding. Plasmid Retention Duration [%] Peak Oxygen Exponential OD.sub.600 CWW before end of Mass Flow Feeding [h] [--] [g/L] induction cultivation [vvm] 7 86 122 100 99 0.2 8 112 184 99 98 0.4 9 136 217 100 98 0.8 10 164 228 99 98 1.5 11 200 262 100 97 >4.5 12 90 186 99 100 >4.5

[0187] According to LDS-PAGE analysis, the specific Qbeta concentration of all cultivations except for the cultivation with 12 h exponential feeding was the same. An optimum regarding absolute Qbeta yield and oxygen consumption was found for 9.5 h exponential feeding. Therefore, the process is preferably run with 9.5 h exponential feeding phase.

Example 20

Scaled Up Fermentation (50 l)

[0188] The process described in Example 6 and with 9.5 h exponential feeding phase with 300 g/L glycerol and 100 g/L lactose as described in Example 19 was scaled up to a volume of 50 L in order to evaluate scale-up capability from the 2 L working volume bioreactor system to a larger volume. Key process parameters for the scaled up process are summarized in Table 11.

TABLE-US-00011 TABLE 11 Process parameters on the 50 L scale Time Culture Step Description [h] Preculture 200 .mu.l from cell bank vial were transferred -18* into 800 ml preculture medium contained in 3000 mL shake flask and cultured for 18 h (2 flasks) Inoculation Pooled total volume (approx. 1.6 L) was 0 of Bioreactor transferred into the 50 L Bioreactor. Initial volume: =35 L Induction The exponential feeding profile was 16.5 Start switched to constant and feed was switched to induction feed End of Culture was completed after 5 h of 21.5 Culture induction *Relative to the time of bioreactor inoculation.

[0189] It was necessary to change the preculture procedure in order to inoculate the larger reactor with approximately the same cell density. The cultures in the 50 L bioreactors were performed with the time profile optimised for the 2 L scale as described in Example 19. The final cell wet weight for six cultivations was 188 g/L.+-.9. Plasmid retention was 97.3%.+-.1.4 at the end of culture. The concentration of Q.beta. CP protein in the medium at the end of culture was determined by C.sub.4 reversed phase HPLC (Example 17) to 10.8 g/L.+-.0.3. The total amount of Q.beta. CP was 540 g for one 50 L run. The crude extract of approximately two times concentrated biomass was analysed for Q.beta. CP and Q.beta. VLP (Example 18). The concentration of Q.beta. CP was 19.1 g/L.+-.0.4 (C.sub.4 reversed phase HPLC), the concentration of Q.beta. VLP was 18.8 g/L.+-.1.1. Therefore, the VLP-yield of the fermentation process is estimated to approximately 9-11 g/l fermentation broth at the time of harvest.

Sequence CWU 1

1

4015579DNAartificial sequenceplasmid 1ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg 60gataatgttt tttgcgccga catcataacg gttctggcaa atattctgaa atgagctgtt 120gacaattaat catcggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca 180ggaaacagaa ttctaaggag gaaaaaaaaa tggcaaaatt agagactgtt actttaggta 240acatcgggaa agatggaaaa caaactctgg tcctcaatcc gcgtggggta aatcccacta 300acggcgttgc ctcgctttca caagcgggtg cagttcctgc gctggagaag cgtgttaccg 360tttcggtatc tcagccttct cgcaatcgta agaactacaa ggtccaggtt aagatccaga 420acccgaccgc ttgcactgca aacggttctt gtgacccatc cgttactcgc caggcatatg 480ctgacgtgac cttttcgttc acgcagtata gtaccgatga ggaacgagct tttgttcgta 540cagagcttgc tgctctgctc gctagtcctc tgctgatcga tgctattgat cagctgaacc 600cagcgtatta atgactgctc attgccggtg gtggctcagg gtcaaaaccc gatccggtta 660ttccggatcc accgattgat ccgccgccag ggacaggtaa gtatacctgt cccttcgcaa 720tttggtccct agaggaggtt tacgagcctc ctactaagaa ccgaccgtgg cctatctata 780atgctgttga actccagcct cgcgaatttg atgttgccct caaagatctt ttgggcaata 840caaagtggcg tgattgggat tctcggctta gttataccac gttccgcggt tgccgtggca 900atggttatat tgaccttgat gcgacttatc ttgctactga tcaggctatg cgtgatcaga 960agtatgatat tcgcgagggc aagaaacctg gtgctttcgg taacattgag cgattcattt 1020atcttaagtc gataaatgct tattgctctc ttagcgatat tgcggcctat cacgccgatg 1080gcgtgatagt tggcttttgg cgcgatccat ccagtggtgg tgccataccg tttgacttca 1140ctaagtttga taagactaaa tgtcctattc aagccgtgat agtcgttcct cgtgcttagt 1200aactaaggat gaaatgcatg tctaagcttg gctgttttgg cggatgagag aagattttca 1260gcctgataca gattaaatca gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg 1320gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa cgccgtagcg 1380ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa 1440cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct 1500ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga 1560gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca gaaggccatc 1620ctgacggatg gcctttttgc gtttctacaa actcttttgt ttatttttct agagccacgt 1680tgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat 1740aaaactgtct gcttacataa acagtaatac aaggagtgtt atgagccata ttcaacggga 1800aacgtcttgc tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa 1860atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc 1920cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga 1980tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt 2040tatccgtact cctgatgatg catggttact caccactgcg atccccggga aaacagcatt 2100ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt 2160cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt 2220tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga 2280tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata agcttttgcc 2340attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga 2400cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca 2460ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct 2520ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct 2580cgatgagttt ttctaaacgc gtgaccaagt ttactcatat gtactttaga ttgatttaaa 2640acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 2700aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 2760atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 2820gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 2880tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 2940ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 3000ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 3060ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 3120aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 3180cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 3240gagggagctc ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 3300ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 3360cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 3420tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 3480cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 3540cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac 3600tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta 3660cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg 3720gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 3780tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca 3840gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt 3900ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt 3960tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata 4020ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta 4080ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc 4140actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag 4200cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc 4260agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt 4320ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta 4380aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt 4440ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat ggcggacgcg 4500atggatatgt tctgccaagg gttggtttgc gcattcacag ttctccgcaa gaattgattg 4560gctccaattc ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc attcaggtcg 4620aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg tatagggcgg 4680cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa atcgccgtga 4740cgatcagcgg tccaatgatc gaagttaggc tggtaagagc cgcgagcgat ccttgaagct 4800gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg ggcatcccga 4860tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc gtcgcgaacg 4920ccagcaagac gtagcccagc gcgtcggccg ccatgccggc gataatggcc tgcttctcgc 4980cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga 5040ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa 5100tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa 5160gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc 5220tcaagggcat cggtcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta 5280gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc 5340ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga 5400gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa 5460ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atccgggctt 5520atcgactgca cggtgcacca atgcttctgg cgtcaggcag ccatcggaag ctgtggtat 55792245DNAartificial sequencepromoter sequence 2cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc atcggaagct gtggtatggc 60tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc cgttctggat 120aatgtttttt gcgccgacat cataacggtt ctggcaaata ttctgaaatg agctgttgac 180aattaatcat cggctcgtat aatgtgtgga attgtgagcg gataacaatt tcacacagga 240aacag 245319DNAartificial sequenceShine-Dalgarno Sequence 3taaggaggaa aaaaaaatg 19418DNAartificial sequenceShine-Dalgarno Sequence 4aggaggtaaa aaacgatg 185133PRTBacteriophage Qbeta 5Met Ala Lys Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Lys Asp Gly1 5 10 15Lys Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly 20 25 30Val Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg 35 40 45Val Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys 50 55 60Val Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser65 70 75 80Cys Asp Pro Ser Val Thr Arg Gln Ala Tyr Ala Asp Val Thr Phe Ser 85 90 95Phe Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg Thr Glu 100 105 110Leu Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln 115 120 125Leu Asn Pro Ala Tyr 13061017DNAartificial sequenceExpression construct 6atggcaaaat tagagactgt tactttaggt aacatcggga aagatggaaa acaaactctg 60gtcctcaatc cgcgtggggt aaatcccact aacggcgttg cctcgctttc acaagcgggt 120gcagttcctg cgctggagaa gcgtgttacc gtttcggtat ctcagccttc tcgcaatcgt 180aagaactaca aggtccaggt taagatccag aacccgaccg cttgcactgc aaacggttct 240tgtgacccat ccgttactcg ccaggcatat gctgacgtga ccttttcgtt cacgcagtat 300agtaccgatg aggaacgagc ttttgttcgt acagagcttg ctgctctgct cgctagtcct 360ctgctgatcg atgctattga tcagctgaac ccagcgtatt aatgactgct cattgccggt 420ggtggctcag ggtcaaaacc cgatccggtt attccggatc caccgattga tccgccgcca 480gggacaggta agtatacctg tcccttcgca atttggtccc tagaggaggt ttacgagcct 540cctactaaga accgaccgtg gcctatctat aatgctgttg aactccagcc tcgcgaattt 600gatgttgccc tcaaagatct tttgggcaat acaaagtggc gtgattggga ttctcggctt 660agttatacca cgttccgcgg ttgccgtggc aatggttata ttgaccttga tgcgacttat 720cttgctactg atcaggctat gcgtgatcag aagtatgata ttcgcgaggg caagaaacct 780ggtgctttcg gtaacattga gcgattcatt tatcttaagt cgataaatgc ttattgctct 840cttagcgata ttgcggccta tcacgccgat ggcgtgatag ttggcttttg gcgcgatcca 900tccagtggtg gtgccatacc gtttgacttc actaagtttg ataagactaa atgtcctatt 960caagccgtga tagtcgttcc tcgtgcttag taactaagga tgaaatgcat gtctaag 10177132PRTBacteriophage Qbeta 7Ala Lys Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Arg Asp Gly Lys1 5 10 15Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val 20 25 30Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg Val 35 40 45Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys Val 50 55 60Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser Cys65 70 75 80Asp Pro Ser Val Thr Arg Gln Lys Tyr Ala Asp Val Thr Phe Ser Phe 85 90 95Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln Leu 115 120 125Asn Pro Ala Tyr 1308132PRTBacteriophage Qbeta 8Ala Lys Leu Glu Thr Val Thr Leu Gly Lys Ile Gly Lys Asp Gly Lys1 5 10 15Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val 20 25 30Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg Val 35 40 45Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys Val 50 55 60Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser Cys65 70 75 80Asp Pro Ser Val Thr Arg Gln Lys Tyr Ala Asp Val Thr Phe Ser Phe 85 90 95Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln Leu 115 120 125Asn Pro Ala Tyr 1309132PRTBacteriophage Qb 9Ala Arg Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Arg Asp Gly Lys1 5 10 15Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val 20 25 30Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg Val 35 40 45Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys Val 50 55 60Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser Cys65 70 75 80Asp Pro Ser Val Thr Arg Gln Lys Tyr Ala Asp Val Thr Phe Ser Phe 85 90 95Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln Leu 115 120 125Asn Pro Ala Tyr 13010132PRTBacteriophage Qbeta 10Ala Lys Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Lys Asp Gly Arg1 5 10 15Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val 20 25 30Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg Val 35 40 45Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys Val 50 55 60Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser Cys65 70 75 80Asp Pro Ser Val Thr Arg Gln Lys Tyr Ala Asp Val Thr Phe Ser Phe 85 90 95Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln Leu 115 120 125Asn Pro Ala Tyr 13011132PRTBacteriophage Qbeta 11Ala Arg Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Lys Asp Gly Arg1 5 10 15Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val 20 25 30Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg Val 35 40 45Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys Val 50 55 60Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser Cys65 70 75 80Asp Pro Ser Val Thr Arg Gln Lys Tyr Ala Asp Val Thr Phe Ser Phe 85 90 95Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln Leu 115 120 125Asn Pro Ala Tyr 13012131PRTBacteriophage AP205 12Met Ala Asn Lys Pro Met Gln Pro Ile Thr Ser Thr Ala Asn Lys Ile1 5 10 15Val Trp Ser Asp Pro Thr Arg Leu Ser Thr Thr Phe Ser Ala Ser Leu 20 25 30Leu Arg Gln Arg Val Lys Val Gly Ile Ala Glu Leu Asn Asn Val Ser 35 40 45Gly Gln Tyr Val Ser Val Tyr Lys Arg Pro Ala Pro Lys Pro Glu Gly 50 55 60Cys Ala Asp Ala Cys Val Ile Met Pro Asn Glu Asn Gln Ser Ile Arg65 70 75 80Thr Val Ile Ser Gly Ser Ala Glu Asn Leu Ala Thr Leu Lys Ala Glu 85 90 95Trp Glu Thr His Lys Arg Asn Val Asp Thr Leu Phe Ala Ser Gly Asn 100 105 110Ala Gly Leu Gly Phe Leu Asp Pro Thr Ala Ala Ile Val Ser Ser Asp 115 120 125Thr Thr Ala 13013131PRTBacteriophage AP205 13Met Ala Asn Lys Thr Met Gln Pro Ile Thr Ser Thr Ala Asn Lys Ile1 5 10 15Val Trp Ser Asp Pro Thr Arg Leu Ser Thr Thr Phe Ser Ala Ser Leu 20 25 30Leu Arg Gln Arg Val Lys Val Gly Ile Ala Glu Leu Asn Asn Val Ser 35 40 45Gly Gln Tyr Val Ser Val Tyr Lys Arg Pro Ala Pro Lys Pro Glu Gly 50 55 60Cys Ala Asp Ala Cys Val Ile Met Pro Asn Glu Asn Gln Ser Ile Arg65 70 75 80Thr Val Ile Ser Gly Ser Ala Glu Asn Leu Ala Thr Leu Lys Ala Glu 85 90 95Trp Glu Thr His Lys Arg Asn Val Asp Thr Leu Phe Ala Ser Gly Asn 100 105 110Ala Gly Leu Gly Phe Leu Asp Pro Thr Ala Ala Ile Val Ser Ser Asp 115 120 125Thr Thr Ala 13014131PRTBacteriophage AP205 14Met Ala Asn Lys Pro Met Gln Pro Ile Thr Ser Thr Ala Asp Lys Ile1 5 10 15Val Trp Ser Asp Pro Thr Arg Leu Ser Thr Thr Phe Ser Ala Ser Leu 20 25 30Leu Arg Gln Arg Val Lys Val Gly Ile Ala Glu Leu Asn Asn Val Ser 35 40 45Gly Gln Tyr Val Ser Val Tyr Lys Arg Pro Ala Pro Lys Pro Glu Gly 50 55 60Cys Ala Asp Ala Cys Val Ile Met Pro Asn Glu Asn Gln Ser Ile Arg65 70 75 80Thr Val Ile Ser Gly Ser Ala Glu Asn Leu Ala Thr Leu Lys Ala Glu 85 90 95Trp Glu Thr His Lys Arg Asn Val Asp Thr Leu Phe Ala Ser Gly Asn 100 105 110Ala Gly Leu Gly Phe Leu Asp Pro Thr Ala Ala Ile Val Ser Ser Asp 115 120 125Thr Thr Ala 13015329PRTBacteriophage Qbeta 15Met Ala Lys Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Lys Asp Gly1 5 10 15Lys Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly 20 25 30Val Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg 35 40 45Val Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys 50 55 60Val Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser65

70 75 80Cys Asp Pro Ser Val Thr Arg Gln Ala Tyr Ala Asp Val Thr Phe Ser 85 90 95Phe Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg Thr Glu 100 105 110Leu Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln 115 120 125Leu Asn Pro Ala Tyr Trp Thr Leu Leu Ile Ala Gly Gly Gly Ser Gly 130 135 140Ser Lys Pro Asp Pro Val Ile Pro Asp Pro Pro Ile Asp Pro Pro Pro145 150 155 160Gly Thr Gly Lys Tyr Thr Cys Pro Phe Ala Ile Trp Ser Leu Glu Glu 165 170 175Val Tyr Glu Pro Pro Thr Lys Asn Arg Pro Trp Pro Ile Tyr Asn Ala 180 185 190Val Glu Leu Gln Pro Arg Glu Phe Asp Val Ala Leu Lys Asp Leu Leu 195 200 205Gly Asn Thr Lys Trp Arg Asp Trp Asp Ser Arg Leu Ser Tyr Thr Thr 210 215 220Phe Arg Gly Cys Arg Gly Asn Gly Tyr Ile Asp Leu Asp Ala Thr Tyr225 230 235 240Leu Ala Thr Asp Gln Ala Met Arg Asp Gln Lys Tyr Asp Ile Arg Glu 245 250 255Gly Lys Lys Pro Gly Ala Phe Gly Asn Ile Glu Arg Phe Ile Tyr Leu 260 265 270Lys Ser Ile Asn Ala Tyr Cys Ser Leu Ser Asp Ile Ala Ala Tyr His 275 280 285Ala Asp Gly Val Ile Val Gly Phe Trp Arg Asp Pro Ser Ser Gly Gly 290 295 300Ala Ile Pro Phe Asp Phe Thr Lys Phe Asp Lys Thr Lys Cys Pro Ile305 310 315 320Gln Ala Val Ile Val Val Pro Arg Ala 32516129PRTBacteriophage R17 16Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asn Asp Gly Gly Thr Gly1 5 10 15Asn Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu Trp 20 25 30Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser Val 35 40 45Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu Val 50 55 60Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val Ala65 70 75 80Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe Ala 85 90 95Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu Leu 100 105 110Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly Ile 115 120 125Tyr17130PRTBacteriophage fr 17Met Ala Ser Asn Phe Glu Glu Phe Val Leu Val Asp Asn Gly Gly Thr1 5 10 15Gly Asp Val Lys Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45Val Arg Gln Ser Ser Ala Asn Asn Arg Lys Tyr Thr Val Lys Val Glu 50 55 60Val Pro Lys Val Ala Thr Gln Val Gln Gly Gly Val Glu Leu Pro Val65 70 75 80Ala Ala Trp Arg Ser Tyr Met Asn Met Glu Leu Thr Ile Pro Val Phe 85 90 95Ala Thr Asn Asp Asp Cys Ala Leu Ile Val Lys Ala Leu Gln Gly Thr 100 105 110Phe Lys Thr Gly Asn Pro Ile Ala Thr Ala Ile Ala Ala Asn Ser Gly 115 120 125Ile Tyr 13018130PRTBacteriophage GA 18Met Ala Thr Leu Arg Ser Phe Val Leu Val Asp Asn Gly Gly Thr Gly1 5 10 15Asn Val Thr Val Val Pro Val Ser Asn Ala Asn Gly Val Ala Glu Trp 20 25 30Leu Ser Asn Asn Ser Arg Ser Gln Ala Tyr Arg Val Thr Ala Ser Tyr 35 40 45Arg Ala Ser Gly Ala Asp Lys Arg Lys Tyr Ala Ile Lys Leu Glu Val 50 55 60Pro Lys Ile Val Thr Gln Val Val Asn Gly Val Glu Leu Pro Gly Ser65 70 75 80Ala Trp Lys Ala Tyr Ala Ser Ile Asp Leu Thr Ile Pro Ile Phe Ala 85 90 95Ala Thr Asp Asp Val Thr Val Ile Ser Lys Ser Leu Ala Gly Leu Phe 100 105 110Lys Val Gly Asn Pro Ile Ala Glu Ala Ile Ser Ser Gln Ser Gly Phe 115 120 125Tyr Ala 13019132PRTBacteriophage SP 19Met Ala Lys Leu Asn Gln Val Thr Leu Ser Lys Ile Gly Lys Asn Gly1 5 10 15Asp Gln Thr Leu Thr Leu Thr Pro Arg Gly Val Asn Pro Thr Asn Gly 20 25 30Val Ala Ser Leu Ser Glu Ala Gly Ala Val Pro Ala Leu Glu Lys Arg 35 40 45Val Thr Val Ser Val Ala Gln Pro Ser Arg Asn Arg Lys Asn Phe Lys 50 55 60Val Gln Ile Lys Leu Gln Asn Pro Thr Ala Cys Thr Arg Asp Ala Cys65 70 75 80Asp Pro Ser Val Thr Arg Ser Ala Phe Ala Asp Val Thr Leu Ser Phe 85 90 95Thr Ser Tyr Ser Thr Asp Glu Glu Arg Ala Leu Ile Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Ala Asp Pro Leu Ile Val Asp Ala Ile Asp Asn Leu 115 120 125Asn Pro Ala Tyr 13020329PRTBacteriophage 20Ala Lys Leu Asn Gln Val Thr Leu Ser Lys Ile Gly Lys Asn Gly Asp1 5 10 15Gln Thr Leu Thr Leu Thr Pro Arg Gly Val Asn Pro Thr Asn Gly Val 20 25 30Ala Ser Leu Ser Glu Ala Gly Ala Val Pro Ala Leu Glu Lys Arg Val 35 40 45Thr Val Ser Val Ala Gln Pro Ser Arg Asn Arg Lys Asn Phe Lys Val 50 55 60Gln Ile Lys Leu Gln Asn Pro Thr Ala Cys Thr Arg Asp Ala Cys Asp65 70 75 80Pro Ser Val Thr Arg Ser Ala Phe Ala Asp Val Thr Leu Ser Phe Thr 85 90 95Ser Tyr Ser Thr Asp Glu Glu Arg Ala Leu Ile Arg Thr Glu Leu Ala 100 105 110Ala Leu Leu Ala Asp Pro Leu Ile Val Asp Ala Ile Asp Asn Leu Asn 115 120 125Pro Ala Tyr Trp Ala Ala Leu Leu Val Ala Ser Ser Gly Gly Gly Asp 130 135 140Asn Pro Ser Asp Pro Asp Val Pro Val Val Pro Asp Val Lys Pro Pro145 150 155 160Asp Gly Thr Gly Arg Tyr Lys Cys Pro Phe Ala Cys Tyr Arg Leu Gly 165 170 175Ser Ile Tyr Glu Val Gly Lys Glu Gly Ser Pro Asp Ile Tyr Glu Arg 180 185 190Gly Asp Glu Val Ser Val Thr Phe Asp Tyr Ala Leu Glu Asp Phe Leu 195 200 205Gly Asn Thr Asn Trp Arg Asn Trp Asp Gln Arg Leu Ser Asp Tyr Asp 210 215 220Ile Ala Asn Arg Arg Arg Cys Arg Gly Asn Gly Tyr Ile Asp Leu Asp225 230 235 240Ala Thr Ala Met Gln Ser Asp Asp Phe Val Leu Ser Gly Arg Tyr Gly 245 250 255Val Arg Lys Val Lys Phe Pro Gly Ala Phe Gly Ser Ile Lys Tyr Leu 260 265 270Leu Asn Ile Gln Gly Asp Ala Trp Leu Asp Leu Ser Glu Val Thr Ala 275 280 285Tyr Arg Ser Tyr Gly Met Val Ile Gly Phe Trp Thr Asp Ser Lys Ser 290 295 300Pro Gln Leu Pro Thr Asp Phe Thr Gln Phe Asn Ser Ala Asn Cys Pro305 310 315 320Val Gln Thr Val Ile Ile Ile Pro Ser 32521130PRTBacteriophage MS2 21Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr1 5 10 15Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50 55 60Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val65 70 75 80Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe 85 90 95Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu 100 105 110Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly 115 120 125Ile Tyr 13022133PRTBacteriophage M11 22Met Ala Lys Leu Gln Ala Ile Thr Leu Ser Gly Ile Gly Lys Lys Gly1 5 10 15Asp Val Thr Leu Asp Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly 20 25 30Val Ala Ala Leu Ser Glu Ala Gly Ala Val Pro Ala Leu Glu Lys Arg 35 40 45Val Thr Ile Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys 50 55 60Val Gln Val Lys Ile Gln Asn Pro Thr Ser Cys Thr Ala Ser Gly Thr65 70 75 80Cys Asp Pro Ser Val Thr Arg Ser Ala Tyr Ser Asp Val Thr Phe Ser 85 90 95Phe Thr Gln Tyr Ser Thr Val Glu Glu Arg Ala Leu Val Arg Thr Glu 100 105 110Leu Gln Ala Leu Leu Ala Asp Pro Met Leu Val Asn Ala Ile Asp Asn 115 120 125Leu Asn Pro Ala Tyr 13023133PRTBacteriophage MX1 23Met Ala Lys Leu Gln Ala Ile Thr Leu Ser Gly Ile Gly Lys Asn Gly1 5 10 15Asp Val Thr Leu Asn Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly 20 25 30Val Ala Ala Leu Ser Glu Ala Gly Ala Val Pro Ala Leu Glu Lys Arg 35 40 45Val Thr Ile Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys 50 55 60Val Gln Val Lys Ile Gln Asn Pro Thr Ser Cys Thr Ala Ser Gly Thr65 70 75 80Cys Asp Pro Ser Val Thr Arg Ser Ala Tyr Ala Asp Val Thr Phe Ser 85 90 95Phe Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Leu Val Arg Thr Glu 100 105 110Leu Lys Ala Leu Leu Ala Asp Pro Met Leu Ile Asp Ala Ile Asp Asn 115 120 125Leu Asn Pro Ala Tyr 13024330PRTBacteriophage NL95 24Met Ala Lys Leu Asn Lys Val Thr Leu Thr Gly Ile Gly Lys Ala Gly1 5 10 15Asn Gln Thr Leu Thr Leu Thr Pro Arg Gly Val Asn Pro Thr Asn Gly 20 25 30Val Ala Ser Leu Ser Glu Ala Gly Ala Val Pro Ala Leu Glu Lys Arg 35 40 45Val Thr Val Ser Val Ala Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys 50 55 60Val Gln Ile Lys Leu Gln Asn Pro Thr Ala Cys Thr Lys Asp Ala Cys65 70 75 80Asp Pro Ser Val Thr Arg Ser Gly Ser Arg Asp Val Thr Leu Ser Phe 85 90 95Thr Ser Tyr Ser Thr Glu Arg Glu Arg Ala Leu Ile Arg Thr Glu Leu 100 105 110Ala Ala Leu Leu Lys Asp Asp Leu Ile Val Asp Ala Ile Asp Asn Leu 115 120 125Asn Pro Ala Tyr Trp Ala Ala Leu Leu Ala Ala Ser Pro Gly Gly Gly 130 135 140Asn Asn Pro Tyr Pro Gly Val Pro Asp Ser Pro Asn Val Lys Pro Pro145 150 155 160Gly Gly Thr Gly Thr Tyr Arg Cys Pro Phe Ala Cys Tyr Arg Arg Gly 165 170 175Glu Leu Ile Thr Glu Ala Lys Asp Gly Ala Cys Ala Leu Tyr Ala Cys 180 185 190Gly Ser Glu Ala Leu Val Glu Phe Glu Tyr Ala Leu Glu Asp Phe Leu 195 200 205Gly Asn Glu Phe Trp Arg Asn Trp Asp Gly Arg Leu Ser Lys Tyr Asp 210 215 220Ile Glu Thr His Arg Arg Cys Arg Gly Asn Gly Tyr Val Asp Leu Asp225 230 235 240Ala Ser Val Met Gln Ser Asp Glu Tyr Val Leu Ser Gly Ala Tyr Asp 245 250 255Val Val Lys Met Gln Pro Pro Gly Thr Phe Asp Ser Pro Arg Tyr Tyr 260 265 270Leu His Leu Met Asp Gly Ile Tyr Val Asp Leu Ala Glu Val Thr Ala 275 280 285Tyr Arg Ser Tyr Gly Met Val Ile Gly Phe Trp Thr Asp Ser Lys Ser 290 295 300Pro Gln Leu Pro Thr Asp Phe Thr Arg Phe Asn Arg His Asn Cys Pro305 310 315 320Val Gln Thr Val Ile Val Ile Pro Ser Leu 325 33025129PRTBacteriophage f2 25Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asn Asp Gly Gly Thr Gly1 5 10 15Asn Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu Trp 20 25 30Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser Val 35 40 45Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu Val 50 55 60Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val Ala65 70 75 80Ala Trp Arg Ser Tyr Leu Asn Leu Glu Leu Thr Ile Pro Ile Phe Ala 85 90 95Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu Leu 100 105 110Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly Ile 115 120 125Tyr26128PRTBacteriophage PP7 26Met Ser Lys Thr Ile Val Leu Ser Val Gly Glu Ala Thr Arg Thr Leu1 5 10 15Thr Glu Ile Gln Ser Thr Ala Asp Arg Gln Ile Phe Glu Glu Lys Val 20 25 30Gly Pro Leu Val Gly Arg Leu Arg Leu Thr Ala Ser Leu Arg Gln Asn 35 40 45Gly Ala Lys Thr Ala Tyr Arg Val Asn Leu Lys Leu Asp Gln Ala Asp 50 55 60Val Val Asp Cys Ser Thr Ser Val Cys Gly Glu Leu Pro Lys Val Arg65 70 75 80Tyr Thr Gln Val Trp Ser His Asp Val Thr Ile Val Ala Asn Ser Thr 85 90 95Glu Ala Ser Arg Lys Ser Leu Tyr Asp Leu Thr Lys Ser Leu Val Ala 100 105 110Thr Ser Gln Val Glu Asp Leu Val Val Asn Leu Val Pro Leu Gly Arg 115 120 125274586DNAartificial sequenceCloning Vector 27ttctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacattata cgagccgatg 60attaattgtc aacagctcat ttcagaatat ttgccagaac cgttatgatg tcggcgcaaa 120aaacattatc cagaacggga gtgcgccttg agcgacacga attatgcagt gatttacgac 180ctgcacagcc ataccacagc ttccgatggc tgcctgacgc cagaagcatt ggtgcaccgt 240gcagtcgata agctccggat cctctacgcc ggacgcatcg tggccggcat caccggcgcc 300acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc 360cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg 420ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc 480ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgaccg 540atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc 600gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg 660ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg 720cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc 780aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccgacgc gctgggctac 840gtcttgctgg cgttcgcgac gcgaggctgg atggccttcc ccattatgat tcttctcgct 900tccggcggca tcgggatgcc cgcgttgcag gccatgctgt ccaggcaggt agatgacgac 960catcagggac agcttcaagg atcgctcgcg gctcttacca gcctaacttc gatcactgga 1020ccgctgatcg tcacggcgat ttatgccgcc tcggcgagca catggaacgg gttggcatgg 1080attgtaggcg ccgccctata ccttgtctgc ctccccgcgt tgcgtcgcgg tgcatggagc 1140cgggccacct cgacctgaat ggaagccggc ggcacctcgc taacggattc accactccaa 1200gaattggagc caatcaattc ttgcggagaa ctgtgaatgc gcaaaccaac ccttggcaga 1260acatatccat cgcgtccgcc atctccagca gccgcacgcg gcgcatctcg ggcagcgttg 1320ggtcctggcc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc 1380ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact 1440gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt 1500cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg 1560caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt 1620gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac 1680aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc 1740gtttcatcgg tatcattacc cccatgaaca gaaattcccc cttacacgga ggcatcaagt 1800gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac 1860gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct 1920tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga 1980aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg 2040gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat 2100gacccagtca cgtagcgata gcggagtgta tactggctta

actatgcggc atcagagcag 2160attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa 2220taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 2280ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 2340gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 2400gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 2460cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 2520ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 2580tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 2640gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 2700tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 2760ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 2820ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 2880ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 2940accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 3000tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 3060cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 3120taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 3180caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 3240gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 3300gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 3360ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 3420attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 3480gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 3540tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 3600agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 3660gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 3720actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 3780tgcccggcgt caacacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 3840attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 3900tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 3960tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 4020aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 4080tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaaa gagtttgtag 4140aaacgcaaaa aggccatccg tcaggatggc cttctgctta atttgatgcc tggcagttta 4200tggcgggcgt cctgcccgcc accctccggg ccgttgcttc gcaacgttca aatccgctcc 4260cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc 4320ccagtctttc gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgcat 4380ggggagaccc cacactacca tcggcgctac ggcgtttcac ttctgagttc ggcatggggt 4440caggtgggac caccgcgcta ctgccgccag gcaaattctg ttttatcaga ccgcttctgc 4500gttctgattt aatctgtatc aggctgaaaa tcttctctca tccgccaaaa cagaagcttg 4560gctgcaggtc gacggatccc cgggaa 458628425DNAartificial sequenceTerminator Sequence 28ctgttttggc ggatgagaga agattttcag cctgatacag attaaatcag aacgcagaag 60cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat 120gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc cccatgcgag 180agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc 240gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg 300atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg 360ccaggcatca aattaagcag aaggccatcc tgacggatgg cctttttgcg tttctacaaa 420ctctt 42529816DNAartificial sequenceResistance gene 29ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat 60accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca 120taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc 180tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac 240tgaatccggt gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca 300gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg 360cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga 420atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata 480ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc 540atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt 600tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa 660caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac 720attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg 780cctcgagcaa gacgtttccc gttgaatatg gctcat 816304963DNAartificial sequencePlasmid 30ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg 60gataatgttt tttgcgccga catcataacg gttctggcaa atattctgaa atgagctgtt 120gacaattaat catcggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca 180ggaaacagaa ttctaaggag gaaaaaaaaa tggcaaataa gccaatgcaa ccgatcacat 240ctacagcaaa taaaattgtg tggtcggatc caactcgttt atcaactaca ttttcagcaa 300gtctgttacg ccaacgtgtt aaagttggta tagccgaact gaataatgtt tcaggtcaat 360atgtatctgt ttataagcgt cctgcaccta aaccggaagg ttgtgcagat gcctgtgtca 420ttatgccgaa tgaaaaccaa tccattcgca cagtgatttc agggtcagcc gaaaacttgg 480ctaccttaaa agcagaatgg gaaactcaca aacgtaacgt tgacacactc ttcgcgagcg 540gcaacgccgg tttgggtttc cttgacccta ctgcggctat cgtatcgtct gatactactg 600cttaatgaag cttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa 660atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt 720cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg 780gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga 840aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa 900atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac 960gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt 1020ttgcgtttct acaaactctt ttgtttattt ttctagagcc acgttgtgtc tcaaaatctc 1080tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac 1140ataaacagta atacaaggag tgttatgagc catattcaac gggaaacgtc ttgctcgagg 1200ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat 1260gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg 1320tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta 1380aactggctga cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat 1440gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt attagaagaa 1500tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat 1560tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg 1620caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc 1680tggcctgttg aacaagtctg gaaagaaatg cataagcttt tgccattctc accggattca 1740gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata 1800ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta 1860tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt 1920attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa 1980acgcgtgacc aagtttactc atatgtactt tagattgatt taaaacttca tttttaattt 2040aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 2100ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 2160ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 2220tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 2280cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 2340gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 2400gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 2460tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 2520ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 2580gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gctcccaggg 2640ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 2700tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 2760ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct 2820gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga 2880acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt 2940ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc 3000tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg 3060ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg 3120catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac 3180cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg 3240attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta 3300atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg 3360atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga 3420ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg 3480gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc 3540agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc 3600agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac 3660ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc 3720ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc 3780ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggccag gacccaacgc 3840tgcccgagat gcgccgcgtg cggctgctgg agatggcgga cgcgatggat atgttctgcc 3900aagggttggt ttgcgcattc acagttctcc gcaagaattg attggctcca attcttggag 3960tggtgaatcc gttagcgagg tgccgccggc ttccattcag gtcgaggtgg cccggctcca 4020tgcaccgcga cgcaacgcgg ggaggcagac aaggtatagg gcggcgccta caatccatgc 4080caacccgttc catgtgctcg ccgaggcggc ataaatcgcc gtgacgatca gcggtccaat 4140gatcgaagtt aggctggtaa gagccgcgag cgatccttga agctgtccct gatggtcgtc 4200atctacctgc ctggacagca tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag 4260aagaatcata atggggaagg ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc 4320cagcgcgtcg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc 4380gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag 4440gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc 4500cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt 4560catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg 4620acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt 4680gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc 4740cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc 4800ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc 4860ggtgatgccg gccacgatgc gtccggcgta gaggatccgg gcttatcgac tgcacggtgc 4920accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tat 4963316DNAartificial sequenceStop Codon 31tgaaca 6326DNAartificial sequenceStop Codon 32taatga 6334525DNAartificial sequencePlasmid 33tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cagacatgca tttcatcctt 420agttactaag cacgaggaac gactatcacg gcttgaatag gacatttagt cttatcaaac 480ttagtgaagt caaacggtat ggcaccacca ctggatggat cgcgccaaaa gccaactatc 540acgccatcgg cgtgataggc cgcaatatcg ctaagagagc aataagcatt tatcgactta 600agataaatga atcgctcaat gttaccgaaa gcaccaggtt tcttgccctc gcgaatatca 660tacttctgat cacgcatagc ctgatcagta gcaagataag tcgcatcaag gtcaatataa 720ccattgccac ggcaaccgcg gaacgtggta taactaagcc gagaatccca atcacgccac 780tttgtattgc ccaaaagatc tttgagggca acatcaaatt cgcgaggctg gagttcaaca 840gcattataga taggccacgg tcggttctta gtaggaggct cgtaaacctc ctctagggac 900caaattgcga agggacaggt atacttacct gtccctggcg gcggatcaat cggtggatcc 960ggaataaccg gatcgggttt tgaccctgag ccaccaccgg caatgagcag tcattaatac 1020gctgggttca gctgatcaat agcatcgatc agcagaggac tagcgagcag agcagcaagc 1080tctgtacgaa caaaagctcg ttcctcatcg gtactatact gcgtgaacga aaaggtcacg 1140tcagcatatg cctggcgagt aacggatggg tcacaagaac cgtttgcagt gcaagcggtc 1200gggttctgga tcttaacctg gaccttgtag ttcttacgat tgcgagaagg ctgagatacc 1260gaaacggtaa cacgcttctc cagcgcagga actgcacccg cttgtgaaag cgaggcaacg 1320ccgttagtgg gatttacccc acgcggattg aggaccagag tttgttttcc atctttcccg 1380atgttaccta aagtaacagt ctctaatttt gccatcgttt tttacctcct tctagagtca 1440ttatggtttt gccatacatc agtatggtgt agcagcactt attataatct ttattgcctc 1500ttaaaactta atccacatca aaactcaaat acttttaacc ccagcgtcct gtaagctctg 1560cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 1620tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 1680tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 1740gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 1800aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 1860ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 1920gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 1980ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 2040ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 2100cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 2160attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 2220ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 2280aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 2340gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 2400tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 2460ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 2520taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 2580atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 2640actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 2700cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 2760agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 2820gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 2880gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 2940gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 3000gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 3060cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtgggg ggggggggcg 3120ctgaggtctg cctcgtgaag aaggtgttgc tgactcatac caggcctgaa tcgccccatc 3180atccagccag aaagtgaggg agccacggtt gatgagagct ttgttgtagg tggaccagtt 3240ggtgattttg aacttttgct ttgccacgga acggtctgcg ttgtcgggaa gatgcgtgat 3300ctgatccttc aactcagcaa aagttcgatt tattcaacaa agccgccgtc ccgtcaagtc 3360agcgtaatgc tctgccagtg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg 3420agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa 3480agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc 3540tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg 3600tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat 3660ggcaaaagct tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca 3720tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga 3780aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg 3840aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg 3900aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata 3960aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca 4020tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg 4080ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat 4140ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctcga gcaagacgtt 4200tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt 4260attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt ttgagacaca 4320acgtggcttt cccccccccc ccattattga agcatttatc agggttattg tctcatgagc 4380ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 4440cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat 4500aggcgtatca cgaggccctt tcgtc 45253456DNAartificial sequencePrimer Sequence 34gcgcgcgaat tcaggaggta aaaaacgatg gcaaaattag agactgttac tttagg 563533DNAartificial sequencePrimer Sequence 35gcatgcaagc ttagacatgc atttcatcct tag 333657DNAartificial sequencePrimer Sequence 36gcgcgcgaat tctaaggagg aaaaaaaaat ggcaaaatta gagactgtta ctttagg 57373914DNAartificial sequenceCloning Vector 37tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ccccggatcc gtcgacctgc 420aggggggggg gggcgctgag gtctgcctcg tgaagaaggt gttgctgact cataccaggc 480ctgaatcgcc ccatcatcca gccagaaagt gagggagcca cggttgatga gagctttgtt 540gtaggtggac cagttggtga ttttgaactt ttgctttgcc acggaacggt ctgcgttgtc 600gggaagatgc gtgatctgat ccttcaactc agcaaaagtt cgatttattc aacaaagccg 660ccgtcccgtc aagtcagcgt aatgctctgc cagtgttaca accaattaac caattctgat 720tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata 780ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat 840aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct 900attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact 960gaatccggtg agaatggcaa aagcttatgc atttctttcc agacttgttc aacaggccag 1020ccattacgct cgtcatcaaa

atcactcgca tcaaccaaac cgttattcat tcgtgattgc 1080gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa 1140tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat 1200tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca 1260tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt 1320agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac 1380aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca 1440ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc 1500ctcgagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg 1560taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag 1620agattttgag acacaacgtg gctttccccc ccccccctgc aggtcgacgg atccggggaa 1680ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 1740caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 1800cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 1860gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 1920ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 1980ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 2040agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 2100taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 2160cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 2220tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 2280gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 2340gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 2400tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 2460gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 2520cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 2580aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 2640tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 2700ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 2760attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 2820ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 2880tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 2940aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 3000acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 3060aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 3120agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 3180ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 3240agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 3300tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 3360tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 3420attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 3480taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 3540aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 3600caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 3660gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 3720cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 3780tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 3840acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac 3900gaggcccttt cgtc 39143857DNAartificial sequencePrimer sequence 38gcgcgcgaat tctaaggagg aaaaaaaaat ggcaaataag ccaatgcaac cgatcac 573944DNAartificial sequencePrimer Sequence 39gcatgcaagc ttcattaagc agtagtatca gacgatacga tagc 44404962DNAartificial sequenceExpression Construct 40ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg 60gataatgttt tttgcgccga catcataacg gttctggcaa atattctgaa atgagctgtt 120gacaattaat catcggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca 180ggaaacagaa ttcaggaggt aaaaaacgat ggcaaataag ccaatgcaac cgatcacatc 240tacagcaaat aaaattgtgt ggtcggatcc aactcgttta tcaactacat tttcagcaag 300tctgttacgc caacgtgtta aagttggtat agccgaactg aataatgttt caggtcaata 360tgtatctgtt tataagcgtc ctgcacctaa accggaaggt tgtgcagatg cctgtgtcat 420tatgccgaat gaaaaccaat ccattcgcac agtgatttca gggtcagccg aaaacttggc 480taccttaaaa gcagaatggg aaactcacaa acgtaacgtt gacacactct tcgcgagcgg 540caacgccggt ttgggtttcc ttgaccctac tgcggctatc gtatcgtctg atactactgc 600ttaatgaagc ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa 660tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc 720ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg 780tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa 840agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa 900tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg 960cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt 1020tgcgtttcta caaactcttt tgtttatttt tctagagcca cgttgtgtct caaaatctct 1080gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca 1140taaacagtaa tacaaggagt gttatgagcc atattcaacg ggaaacgtct tgctcgaggc 1200cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg 1260tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt 1320ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa 1380actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg 1440atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat 1500atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt 1560cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc 1620aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct 1680ggcctgttga acaagtctgg aaagaaatgc ataagctttt gccattctca ccggattcag 1740tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag 1800gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat 1860ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta 1920ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaaa 1980cgcgtgacca agtttactca tatgtacttt agattgattt aaaacttcat ttttaattta 2040aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 2100tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 2160tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 2220gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 2280agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 2340tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 2400ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 2460cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 2520tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 2580acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag ctcccagggg 2640gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 2700ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 2760tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 2820attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 2880cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc 2940tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct 3000ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc 3060tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc 3120atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc 3180gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga 3240ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa 3300tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga 3360tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag 3420gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg 3480taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca 3540gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca 3600gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg 3660gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct 3720tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc 3780tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct 3840gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca 3900agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt 3960ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat 4020gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc 4080aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccaatg 4140atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca 4200tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga 4260agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc 4320agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg 4380ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg 4440ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc 4500ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc 4560atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga 4620cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg 4680agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc 4740acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc 4800cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg 4860gtgatgccgg ccacgatgcg tccggcgtag aggatccggg cttatcgact gcacggtgca 4920ccaatgcttc tggcgtcagg cagccatcgg aagctgtggt at 4962

Claims

1. A process for expression of a recombinant capsid protein of a RNA bacteriophage being capable of forming a virus-like particle (VLP) by self-assembly, wherein said RNA bacteriophage is RNA bacteriophage Q.beta., and wherein said recombinant capsid protein comprises SEQ ID NO:5, said process comprising: a.) introducing an expression plasmid into a bacterial host, wherein said expression plasmid comprises an expression construct, wherein said expression construct comprises (i) a first nucleotide sequence encoding said recombinant capsid protein, or mutant or fragment thereof, and (ii) a promoter being inducible by lactose; b.) initiating a growth phase by cultivating said bacterial host in a medium comprising a major carbon source; wherein said cultivating initiates a batch phase during said growth phase, wherein said cultivating is performed in batch culture and under conditions under which said promoter is repressed by lacI, wherein said lacI is overexpressed by said bacterial host, and wherein no feeding of said batch culture is performed during said cultivating; c.) ending said batch phase and initiating a feed phase during said growth phase by feeding said batch culture with said major carbon source; wherein said feeding of said batch culture is performed with a flow rate, wherein said flow rate increases with an exponential coefficient .mu., and d.) ending said growth phase and initiating a production phase by inducing said promoter with an inducer, wherein said feeding of said batch culture with said major carbon source is continued; and wherein during said steps (b.), (c.) and (d.) no removal of medium, except for analytical purposes, takes place leading to an increased density of said bacterial host in said medium.

2-5. (canceled)

6. The process of claim 1, wherein said expression construct comprises a first stop codon and a second stop codon, wherein said first stop codon is located directly 3' of said first nucleotide sequence and wherein said second stop codon is located directly 3' of said first stop codon, and wherein at least one of said first or second stop codon is TAA.

7. The process of claim 1, wherein said expression construct further comprises a second nucleotide sequence, wherein said first nucleotide sequence encodes Q.beta. coat protein (CP), and wherein said second nucleotide sequence encodes Q.beta. A1 protein and wherein said first and said second nucleotide sequence are separated by exactly one sequence stretch comprising at least one TAA stop codon.

8. The process of claim 1, wherein said expression construct comprises SEQ ID NO:6.

9. The process of claim 1, wherein said expression plasmid comprises SEQ ID NO:1.

10. (canceled)

11. The process of claim 1, wherein said promoter is selected from the group consisting of a.) tac promoter; b.) trc promoter; c.) tic promoter; d.) lac promoter; e.) lacUV5 promoter; f.) P.sub.syn promoter; g.) lpp.sup.a promoter; h.) lpp-lac promoter; i.) T7-lac promoter; j.) T3-lac promoter; k.) T5-lac promoter; and l.) a promoter having at least 50% sequence homology to SEQ ID NO:2.

12. The process of claim 1, wherein said promoter comprises SEQ ID NO:2.

13. The process of claim 1, wherein said major carbon source is glycerol.

14. The process of claim 1, wherein said exponential coefficient .mu. is below .mu..sub.max.

15. The process of claim 1, wherein said inducing of said promoter is performed by co-feeding said batch culture with said inducer and said major carbon source at a constant flow rate.

16. The process of claim 1, wherein said inducing of said promoter is performed by co-feeding said batch culture with said inducer and said major carbon source at an increasing flow rate.

17. The process of claim 15, wherein said inducer is lactose and wherein said lactose and said major carbon source are co-fed to said batch culture in a ratio of about 2:1 to 1:4 (w/w).

18. The process of claim 1, wherein said inducer is IPTG and wherein the concentration of said IPTG in said medium is 0.001 to 5 mM.

19. (canceled)

20. The process of claim 1, wherein said lacI is overexpressed by said bacterial host, wherein said overexpression is caused by lacI.sup.q or lacQ1.

21. (canceled)

22. (canceled)

23. The process of claim 1, wherein said inducer is lactose and wherein said bacterial host comprises .beta.-galactosidase activity.

24. The process of claim 1, wherein said cultivating and said feeding of said batch culture and said inducing of said promoter is performed at a temperature which is below the optimal growth temperature of said bacterial host.

25. The process of claim 1 wherein: a.) said major carbon source is glycerol; b.) said inducer is lactose; c.) and said lactose and said major carbon source are co-fed to said batch culture in a ratio of 2:1 to 1:4 (w/w); d.) said bacterial host is E. coli RB791; and e.) said cultivating and feeding of said batch culture and said inducing of said promoter is performed at a temperature of about 30.degree. C.

26. (canceled)

27. The process of claim 1, wherein throughout steps b.) to d.) of said process oxygen is supplied to said bacterial host, wherein said oxygen supply is effected such that the partial pressure of oxygen in the medium (pO.sub.2) is at least about 40%.

28. The process of claim 16, wherein said inducer is lactose and wherein said lactose and said major carbon source are co-fed to said batch culture in a ratio of about 2:1 to 1:4 (w/w).

29. The process of claim 1, wherein said inducer is lactose.

30. The process of claim 20, wherein said overexpression is caused by lacI.sup.q.

31. The process of claim 29, wherein said lactose and said major carbon source are co-fed to said batch culture in a ratio of 1:1 to 1:3 (w/w).

32. The process of claim 29, wherein said lactose and said major carbon source are co-fed to said batch culture in a ratio of 1:3 (w/w).