HK1115156B - Rhamnose promoter expression system and use thereof - Google Patents

Description

Rhamnose promoter expression system and application

The present invention relates to vectors for the heterologous expression of nucleic acids encoding, for example, polypeptides (e.g., recombinant proteins) in prokaryotic hosts. More particularly, the present invention relates to a novel vector expressible in a host comprising the rhaBAD promoter region of the L-rhamnose operon operably linked to a transcriptional unit comprising: a) a nucleic acid sequence which is heterologous to said host, b) a prokaryotic signal sequence operatively linked to said nucleic acid sequence, said prokaryotic signal sequence being selected from signal peptides of periplasmatic binding proteins for sugars, amino acids, vitamins and ions. The invention also relates to the use of these vectors for the heterologous expression of nucleic acids encoding, for example, polypeptides.

Background

A number of systems have been described for the heterologous expression of nucleic acids encoding, for example, polypeptides (e.g., recombinant proteins) in prokaryotic hosts. However, most heterologous gene expression systems in prokaryotic host systems rely entirely on a limited set of bacterial promoters. The most commonly used prokaryotic promoters include the lactose [ lac ] (Yanisch-Perron et al, 1985, Gene33, 103-109), and tryptophan [ trp ] (Goeddel et al, 1980, Nature (London) 287, 411-416) promoters, and hybrid promoters [ tac and trc ] (Brosius, 1984, Gene 27: 161-172; Amann and Brosius, 1985, Gene40, 183-190) derived from both promoters. Other inducible promoter systems, such as the araB promoter inducible by arabinose (WO8604356), the rhamnose promoter rhaSB inducible by L-rhamnose (WO03068956) or the rhamnose promoter rhaBAD inducible by L-rhamnose (WO2004/050877) have also been described for heterologous expression of proteins. WO2004/050877 describes the heterologous expression of nitrilases in E.coli using the rhaBAD promoter. Nitrilase activity can be obtained in resting cell assays after induction with L-rhamnose. However, especially in the expression of complex polypeptides (such as antibodies or antibody fragments), it is advantageous to use a signal peptide to transfer the polypeptide from the cytoplasm to a non-cytoplasmic location (secretion), since overproduction of heterologous proteins in the cytoplasm is often accompanied by misfolding and segregation into insoluble aggregates (endosomes). However, since the signal sequence may influence the formation of secondary and tertiary structures in the mature region of the secreted polypeptide, the selection of an appropriate signal peptide and a useful promoter is important for high yield of functional polypeptide. Thus, there remains a need to provide alternative prokaryotic expression systems for heterologous expression of nucleic acid sequences.

Disclosure of Invention

These and other objects, which will be apparent from the foregoing description, are achieved by providing a novel vector which can be used for high level expression of a desired heterologous product, which comprises the rhaBAD promoter region of the L-rhamnose operon, a heterologous nucleic acid sequence and a prokaryotic signal sequence selected from signal peptides of periplasmatic binding proteins for sugars, amino acids, vitamins and ions. In a first aspect, the object of the present invention is to provide a novel vector expressible in a host comprising the rhaBAD promoter region of the L-rhamnose operon operably linked to a transcriptional unit comprising: a) a nucleic acid sequence which is heterologous to said host, b) a prokaryotic signal sequence operatively linked to said nucleic acid sequence, said prokaryotic signal sequence being selected from the group consisting of signal peptides of periplasmatic binding proteins for sugars, amino acids, vitamins and ions, the expression of which is controlled by the promoter. Also provided are: the use of the novel vectors for the modulated heterologous expression of nucleic acid sequences in a host of an original organism; an isolated and purified nucleic acid sequence expressible in a host comprising the rhaBAD promoter region of the L-rhamnose operon, a heterologous nucleic acid sequence and a prokaryotic signal sequence selected from signal peptides of periplasmatic binding proteins for sugars, amino acids, vitamins and ions; a prokaryotic host transformed with said vector or said isolated and purified nucleic acid sequence; a method for producing a polypeptide in a host using the vector; and a vector comprising: a promoter region, a heterologous nucleic acid sequence and a translation initiation region consisting of the sequence AGGAGATATACAT.

Other objects and advantages will become apparent to those skilled in the art from the following detailed description, which proceeds with reference to the following illustrative drawings and the appended claims.

Brief Description of Drawings

FIG. 1 shows plasmid pBW22-Fab-H containing the L-rhamnose inducible promoter (PrhaBAD), sequences encoding the signal sequences operably linked to the light chain (ompA-VL3-CL) and the heavy chain (phoA-VH-CH) of the Fab fragment, and a transcription termination region (rrnB).

FIG. 2 shows plasmid pBLL15, which contains the melibiose inducible promoter (PmelAB2), sequences encoding the signal sequences operably linked to the light chain (ompA-VL3-CL) and the heavy chain (phoA-VH-CH) of the Fab fragment, and the transcription termination region (rrnB).

FIG. 3 shows dot blot results of lysozyme extracts of the uninduced (-) and induced (+) W3110 strains using different expression plasmids (Fab detected with anti-human light chain, alkaline peroxidase conjugated). The time interval is displayed.

FIG. 4 shows plasmid pAKL14 containing the L-rhamnose inducible promoter (PrhaBAD) and the Fab-H gene with altered signal sequence.

FIG. 5 shows dot blots of lysozyme extracts of the uninduced (-and L-rhamnose induced strains W3110(pAKL 14). The sampling time is shown (detection of Fab with anti-human light chain, alkaline peroxidase coupled).

FIG. 6 shows a Western blot of lysozyme extracts of L-rhamnose-induced strain W3110(pAKL 14). The post-induction time at sampling (detection of Fab with anti-human light chain, coupled with alkaline peroxidase) is shown. Lane 1: standard (1.28 μ g); lane 2: w3110(pAKL14), induction, 3 h; lane 3: w3110(pAKL14), induction, 5 h; lane 4: w3110(pAKL14), induction, 7 h; lane 5: w3110(pAKL14), induction, 12 h; lane 6: w3110(pAKL14), induction, 23 h; lane 7: w3110(pAKL14), not induced, 23 h.

FIG. 7 shows SDS-PAGE of lysozyme extracts of different W3110 strains with high Fab-H antibody concentrations. The strain producing the light and heavy chains without signal sequences was used as negative reference (lane 1: marker; lane 2: W3110(pMx 9-HuCAL-Fab-H); lane 3: W3110(pBW 22-Fab-H); lane 4: W3110(pBLL 15); lane 5: W3110(pAKL 14); lane 6: standard (2. mu.g)).

FIG. 8 shows plasmid pAKL15E containing the melibiose inducible promoter (PmelAB2) and the Fab-H gene with altered signal sequence.

FIG. 9 shows SDS-PAGE of lysozyme extracts of strain W3110(pAKL15E) in the presence or absence of the inducer melibiose. The positions of the light and heavy chains are shown (lane 1: marker; lane 2: W3110(pAKL15E), not induced; lane 3: W3110(pAKL15E), induced).

FIG. 10 shows plasmid pBLL7-pelB-S1, which contains the L-rhamnose inducible rhaBAD promoter, a sequence encoding a PelB signal peptide operably linked to a sequence encoding a single chain antibody (scFv, S1), and a transcription termination region (rrnB).

FIG. 11 shows SDS-PAGE of crude extracts of the uninduced (-) and induced (+) W3110(pBW22-pelB-S1) strains. Samples were taken at different time intervals as indicated. The soluble and insoluble protein fractions obtained after lysozyme treatment were analyzed. Arrows indicate scFv proteins. Marker 12(Mark12), molecular weight standard of Invitrogen.

FIG. 12 shows a broad-host range plasmid pJOE4782 containing the L-rhamnose inducible rhaBAD promoter and the genes for the regulatory proteins RhaS and RhaR of the L-rhamnose operon of Escherichia coli. Plasmid pJOE4782 also contains a sequence encoding a MalE signal peptide operably linked to a sequence encoding a GFP reporter protein.

FIG. 13 shows plasmid pAKLP2 containing the L-rhamnose inducible rhaBAD promoter and the sequence encoding the nitrilase protein (nitA).

FIG. 14 shows SDS-PAGE of induced Pseudomonas putida KT2440(Pseudomonas putida strain KT2440) cells (pAKLP 2). Samples were taken after different time intervals as indicated. The arrow indicates the nitrilase protein. Marker 12, molecular weight standard of Invitrogen.

FIG. 15 shows plasmid pAKLP1 containing the L-rhamnose inducible rhaBAD promoter and sequences encoding the Fab-M heavy and light chains operably linked to the sequence encoding the OmpA signal peptide and the sequence encoding the PhoA signal peptide, respectively.

FIG. 16 shows SDS-PAGE of induced Pseudomonas putida KT2440(Pseudomonas putida strain KT2440) cells (pAKLP 1). Samples were taken after different time intervals as indicated. Arrows indicate FabM light and heavy chains. Marker 12, molecular weight standard of Invitrogen.

FIG. 17 shows SDS-PAGE of fermentation samples of E.coli strain W3110(pBW 22-pelB-S1). Samples (in hours) were taken after different time intervals as indicated. Arrows indicate scFv proteins. Marker 12, molecular weight standard of Invitrogen.

Detailed Description

The following definitions are provided herein to facilitate understanding of the invention.

"vector expressible in a host" or "expression vector" refers to a recombinantly produced or synthetic polynucleic acid construct having a series of specified polynucleic acid elements which permit transcription of a particular nucleic acid sequence in a host cell. Generally, such vectors include a transcription unit comprising a particular nucleic acid to be transcribed operably linked to a promoter. Vectors that can be expressed in a host may be, for example: autonomously or self-replicating plasmids, cosmids, phages, viruses or retroviruses.

The terms "host", "host cell" or "recombinant host cell" are used interchangeably herein and refer to a prokaryotic cell into which one or more vectors or isolated and purified nucleic acid sequences of the present invention have been introduced. It is understood that these terms refer not only to the particular cell object, but also to the progeny or potential progeny of such a cell. Because subsequent generations may undergo some alteration due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

The term "comprising" is generally used in the sense of "comprising", i.e. allowing the presence of one or more features or components.

"promoter" as used herein refers to a nucleic acid sequence that regulates the expression of a transcriptional unit. A "promoter region" is a regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. In the promoter region there will be a transcription initiation site (usually defined by mapping with nuclease S1) and protein binding regions (consensus sequences) responsible for the binding of RNA polymerase, such as the putative-35 region and the Pribnow box.

"L-rhamnose operon" refers to the rhaSR-rhaBAD operon described by Holcroft and Egan (2000, J.Bacteriol.182(23), 6774-6782). The RhaBAD operon is a positively regulated catabolic operon that transcribes RhaB, RhaA and RhaD separately from another rha operon, rhaSR, with approximately 240bp of DNA separating them between their respective transcription start sites. The rhaSR promoter encodes the two L-rhamnose-specific activators RhaS and RhaR. RhaR regulates transcription of rhaSR, while RhaS binds to DNA upstream of-32 to-81 relative to the transcription start site of rhaBAD. In addition, the rhaSR-rhaBAD intergenic operon contains a CRP binding site (CRP1) at-92, 5 (relative to the transcription start site of rhaBAD), and CRP binding sites at-92, 5(CRP2), -115, 5(CRP3), 116, 5(CRP4) (relative to the transcription start site of rhaSR), and a CRP binding site of RhaR spanning-32 to-82 (relative to the transcription start site of rhaSR).

"the rhaBAD promoter region of the L-rhamnose operon" means a rhaBAD operon consisting essentially of the rhaBAD transcription initiation site, the putative-35 region, the Pribnow box, the CRP binding site CRP1, the RhaS binding site relative to the transcription initiation site of rhaBAD and the CRP binding site CRP2-4, and the RhaR binding site relative to the transcription initiation site of rhaSR. "rhaBAD promoter" refers to a promoter of the rhaBAD operon consisting essentially of the rhaBAD transcription start site, the putative-35 region, the Pribnow box, the RhaS binding site and CRP1 binding site region relative to the rhaBAD transcription start site, and the CRP binding site CRP4 or a portion thereof relative to the rhaSR transcription start site.

"CRP" means a "catabolite regulator protein". "CRP" is commonly referred to in the art as "cyclic AMP receptor protein" and has the same meaning. CRP is a regulatory protein controlled by cyclic AMP (cAMP) which mediates the activation of catabolic operons such as the L-rhamnose operon.

An "enhancer" is a nucleic acid sequence that enhances transcription of a transcriptional unit independent of the identity of the transcriptional unit, the position of its sequence relative to the transcriptional unit, or the orientation of its sequence. The vectors of the invention optionally contain an enhancer.

"transcriptional unit" as used herein refers to a nucleic acid sequence that is generally transcribed into a single RNA molecule. The transcriptional unit may contain one gene (monocistronic) or two (dicistronic) or more genes (polycistronic) that encode functionally related polypeptide molecules.

A nucleic acid sequence is "operably linked" when it is functionally related to another nucleic acid sequence. For example, if the DNA of the signal sequence is expressed as a precursor protein involved in the secretion of the protein, the DNA of the signal sequence is operably linked to the DNA of the protein; a promoter is operably linked to a sequence if it affects the transcription of the sequence; or a nucleic acid sequence encoding a polypeptide is operably linked if a translation initiation region (e.g., a ribosome binding site) is positioned such that translation of the polypeptide is facilitated. Ligation can be achieved by ligation at conventional restriction sites. If such sites are not present, synthetic oligonucleotide linkers or linkers are used according to conventional practice.

A "nucleic acid" or "nucleic acid sequence" or "isolated and purified nucleic acid or nucleic acid sequence" may be DNA, RNA, or DNA/RNA hybrids in the context of the present invention. In the case where the nucleic acid or nucleic acid sequence is located in a vector, the nucleic acid or nucleic acid sequence is typically DNA. The DNA herein may be any polydeoxynucleotide sequence including, for example: double-stranded DNA, single-stranded DNA, double-stranded DNA wherein one or both strands are composed of two or more fragments, double-stranded DNA wherein one or both strands contain an uninterrupted phosphodiester backbone, DNA comprising one or more single-stranded portions and one or more double-stranded portions, double-stranded DNA wherein the DNA strands are fully complementary, double-stranded DNA wherein the DNA strands are only partially complementary, circular DNA, covalently closed DNA, linear DNA, covalently cross-linked DNA, cDNA, chemically synthesized DNA, semi-synthesized DNA, biosynthetic DNA, naturally-isolated DNA, enzymatically digested DNA, sheared DNA, labeled DNA (e.g., radiolabeled DNA and fluorochrome-labeled DNA), DNA comprising one or more non-naturally-occurring nucleic acid species. The DNA sequence may be synthesized using standard chemical techniques (e.g., the phosphotriester method) or by automated synthesis methods and PCR methods. Purified and isolated DNA can also be produced using enzymatic techniques.

RNA herein may be for example: single-stranded RNA, cRNA, double-stranded RNA wherein one or both strands are composed of two or more fragments, double-stranded RNA wherein one or both strands have an uninterrupted phosphodiester backbone, RNA comprising one or more single-stranded portions and one or more double-stranded portions, double-stranded RNA wherein the RNA strands are fully complementary, double-stranded RNA wherein the RNA strands are only partially complementary, covalently cross-linked RNA, enzymatically digested RNA, sheared RNA, mRNA, chemically synthesized RNA, semi-synthesized RNA, biosynthetic RNA, naturally isolated RNA, labeled RNA (e.g., radiolabeled RNA and fluorochrome-labeled RNA), RNA comprising one or more non-naturally occurring nucleic acid species.

"variant" or "variant of a sequence" refers to a nucleic acid sequence that has conservative nucleic acid substitutions as compared to a reference sequence, such that one or more nucleic acids are substituted for other nucleic acids having the same properties. Variants also include degenerate sequences, sequences with deletions and insertions, as long as the modified sequence has the same function as the reference sequence (functionally equivalent).

As used herein, the terms "polypeptide", "peptide", "protein", "polypeptidic" and "peptidic" are used interchangeably and refer to a series of amino acid residues linked to another amino acid residue by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues.

The term "isolated and purified nucleic acid sequence" refers to the state which a nucleic acid sequence according to the invention will assume. The nucleic acid sequence will be free or substantially free of: substances to which they are naturally associated, such as other nucleic acids to which they are associated as found in their natural environment or in their environment of preparation (e.g., cell culture) when such preparations are made using recombinant techniques, in vitro or in vivo.

The terms "transformation", "transformed" or "introducing a nucleic acid into a host cell" refer to any process by which an extracellular nucleic acid (e.g., a vector) enters a host cell, with or without accompanying material. The term "cell transformed" or "transformed cell" refers to a cell or its progeny into which an extracellular nucleic acid has been introduced so as to contain the extracellular nucleic acid. The nucleic acid may be introduced into the cell such that the nucleic acid is replicable as a chromosomal integrant or as an extra chromosomal element. Suitable host cells can be transformed with, for example, expression vectors using known methods such as microinjection, electroporation, particle bombardment or using chemical methods such as calcium phosphate-mediated transformation (Maniatis et al, 1982, "Molecular cloning, A laboratory Manual," Molecular cloning, Cold Spring harbor laboratory, or Ausubel et al, 1994, "Current Protocols in Molecular Biology," recent advances in Molecular Biology, John Wiley and Sons).

"heterologous nucleic acid sequence" or "nucleic acid sequence heterologous to the host" refers to a nucleic acid sequence encoding an expression product, e.g., a polypeptide, which is foreign to the host ("heterologous expression" or "heterologous product"), i.e., a nucleic acid sequence derived from a donor different from the host, or a chemically synthesized nucleic acid sequence encoding an expression product, e.g., a polypeptide, which is foreign to the host. In case the host is a specific prokaryotic species, the heterologous nucleic acid sequence is preferably derived from a different genus or family, more preferably from a different order or class, especially from a different phylum (division), more especially from a different domain (empire) of organisms.

A heterologous nucleic acid sequence derived from a donor different from the host may be modified by mutation, insertion, deletion or substitution of a single nucleic acid or a portion of the heterologous nucleic acid sequence prior to introduction into the host cell, provided that such modified sequence still has the same function (functional equivalent) as the reference sequence. Heterologous nucleic acid sequences as referred to herein also include nucleic acid sequences derived from organisms of different domains (empire), such as from eukaryotes (of eukaryotic origin), such as human antibodies, which have been used in phage display libraries and have been modified from individual nucleic acids or portions of nucleic acid sequences according to the "codon usage" of a prokaryotic host.

"Signal sequence" or "signal peptide sequence" refers to a nucleic acid sequence encoding a short amino acid sequence (i.e., signal peptide) that is present at the NH 2-terminus of certain proteins and is normally transported by cells to a non-cytoplasmic location (e.g., secretion) or becomes a membrane component. The signal peptide directs the transport of the protein from the cytoplasm to a non-cytoplasmic location.

"translation initiation region" refers to a signal region which promotes translation initiation and serves as a ribosome binding site, such as the Shine Dalgarno sequence.

"transcription termination region" refers to a sequence that causes RNA polymerase to terminate transcription. Transcription termination sequences are usually part of a transcriptional unit, which increases the stability of the mRNA.

"antibody" refers to a class of plasma proteins produced by the B cells of the immune system following antigen stimulation. Mammalian (i.e., human) antibodies are immunoglobulins of the IgG, M, A, E or D class. For the purposes of the present invention, the term "antibody" includes, but is not limited to: polyclonal antibodies, monoclonal antibodies, anti-idiotypic antibodies and autoantibodies present in autoimmune diseases such as diabetes, multiple sclerosis and rheumatoid arthritis, and chimeric antibodies. The basic antibody building block is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain comprises a variable region of about 100-110 or more amino acids, primarily responsible for antigen recognition. The carboxy terminus of each chain defines a constant region, primarily responsible for effector function.

Light chains are divided into K and λ. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and the antibody isotypes are defined as IgG, IgM, IgA, IgD, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids.

The term "antibody" refers to whole antibodies or binding fragments thereof. Binding fragments include single chain fragments, Fv fragments and Fab fragments.

The term "single chain antibody" includes non-native antibody forms that combine only the antigen-binding regions of an antibody on a single stably folded polypeptide chain. Thus, single chain antibodies are much smaller in size than typical immunoglobulins, but they still retain the antigen-specific binding properties of the antibody. Single chain antibodies are widely used in a number of different applications, including, for example, therapeutic, diagnostic, research tools, and the like.

The term Fab fragment sometimes refers in the art to the binding fragment resulting from papain cleavage of an intact antibody. The terms Fab 'and F (ab') 2 sometimes refer in the art to binding fragments produced by pepsin cleavage of an intact antibody. In the context of the present invention, Fab generally refers to a double-chain binding fragment of an intact antibody having at least substantially intact light and heavy chain variable regions sufficient for antigen-specific binding, and portions of the light and heavy chain constant regions sufficient to maintain binding of the light and heavy chains. An example of such a Fab is described, for example, in Skerra et al (1988, Science240(4855), 1038-41). Fab fragments such as IgG idiotypes may or may not contain at least one of the two cysteine residues that form the two interchain disulfide bonds between the two heavy chains in an intact immunoglobulin. Typically, Fab fragments are formed by complexing a full-length or substantially full-length light chain with a heavy chain containing the variable and constant regions, at least the CH1 regions. In addition, the C-terminal cysteine on the light chain may be replaced with serine or other amino acids to eliminate the interchain disulfide bond between the light and heavy chains of the present invention.

Also included are chimeric antibodies in which the light and heavy chain genes are constructed, for example, from immunoglobulin gene segments (e.g., segments encoding variable regions and segments encoding constant regions) of different species, typically using genetic engineering techniques. For example, the variable (V) segments of the mouse monoclonal antibody gene can be linked to human constant (C) segments (e.g., IgG1 and IgG 4). Thus, a typical chimeric antibody is a hybrid protein consisting of the V or antigen binding domain of a mouse antibody and the C or effector domain of a human antibody. The binding specificity and affinity of a chimeric antibody is the same or similar to that of a mouse antibody or other non-human antibody that provides the variable region of the antibody.

The term "human antibody" includes antibodies having variable and constant regions (if present) derived from human germline (germline) immunoglobulin sequences including natural or artificial, engineered affinity maturation. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does include antibodies (i.e., humanized antibodies) that have been grafted onto human framework sequences derived from CDR sequences of other mammalian germline, e.g., mouse. The term also includes functional variants of these "human antibodies", such as truncated forms thereof, or engineered variants in which, for example, a single proline or cysteine residue has been engineered by genetic engineering techniques known in the art. Examples of these are described in WO 98/02462. However, the term relates only to the amino acid sequences of these antibodies, without regard to any glycosylation or other chemical modification of the peptide backbone.

In one aspect, the present invention provides a vector expressible in a host comprising the rhaBAD promoter region of the L-rhamnose operon operably linked to a transcriptional unit comprising

a) A nucleic acid sequence which is heterologous to the host,

b) a prokaryotic signal sequence operably linked to said nucleic acid sequence, said prokaryotic signal sequence being selected from the group consisting of signal peptides of periplasmatic binding proteins for sugars, amino acids, vitamins and ions, and the expression of said nucleic acid sequence being controlled by said promoter region.

The vector of the invention is preferably an autonomously or self-replicating plasmid, cosmid, phage, virus or retrovirus. A wide range of host/vector combinations may be used to express the nucleic acid sequences of the present invention. Useful expression vectors can be comprised of segments of, for example, chromosomal, nonchromosomal, and/or synthetic nucleic acid sequences. Suitable vectors include vectors with a specific host range, e.g.specific for e.g.E.coli, as well as vectors with a broad host range, e.g.for gram-negative bacteria. "Low copy", "medium copy" and "high copy" plasmids may be used.

Useful vectors for expression in e.g.E.coli are: pQE70, pQE60 and pQE-9(QIAGEN, Inc.); pBluescript Vektoren, Phagescript Vektoren, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene Cloning Systems, Inc.); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia Bio-tech, Inc.); pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pACYC177, pACYC184, pRSF1010 and pBW22[ Wilms et al, 2001, Biotechnology and Bioengineering, 73(2)95-103] or derivatives thereof, such as plasmid pBW-22-Fab-H or pAKL 14. Other useful plasmids are well known to those skilled in the art and are described, for example, in "Cloning Vectors" ("Cloning Vectors" edited by Pouwels P.H., et al, Elsevier, Amsterdam-New York-Oxford, 1985).

Preferred vectors of the invention are autonomously or self-replicating plasmids, more preferably vectors with a specific host range, e.g.vectors specific for e.g.E.coli. Most preferred are pBR322, pUC18, pACYC177, pACYC184, pRSF1010 and pBW22 or derivatives thereof, such as pBW22-Fab-H or pAKL14, especially preferred are pBW22-Fab-H or pAKL14, most preferred is pAKL 14.

In a preferred embodiment, the rhaBAD promoter region of the L-rhamnose operon is the rhaBAD promoter. In a particularly preferred embodiment, the rhaBAD promoter consists of SEQ ID NO: 1. the complementary sequence or variant thereof. Preferably, the rhaBAD promoter region of the L-rhamnose operon, the rhaBAD promoter and the promoter sequence consisting of SEQ ID NO: 1. the rhaBAD promoter consisting of the complementary sequence or variant thereof is derived from the L-rhamnose operon of Escherichia coli.

In another preferred embodiment of the invention, the host expressible in a prokaryotic host comprises, in addition to the rhaBAD promoter region of the L-rhamnose operon operably linked to a transcriptional unit, sequences encoding the L-rhamnose specific activators RhaS and RhaR (including their respective native promoter sequences). Upon expression, the RhaS and RhaR proteins control the activity of the rhaBAD promoter.

For prokaryotic signal sequences of signal peptides of periplasmatic binding proteins selected from sugars, amino acids, vitamins and ions, for example: PelB (Erwinia chrysanthemi (Erwinia chrysanate), pectate lyase precursor), PelB (Erwinia carotovora, pectate lyase precursor), PelB (Xanthomonas campestris (xanthmonas campestris), pectate lyase precursor), LamB (escherichia coli, maltoporin precursor), MalE (escherichia coli, maltose binding protein precursor), Bla (escherichia coli, beta-lactamase), OppA (escherichia coli, periplasmic oligopeptide-binding protein), TreA (escherichia coli, periplasmic trehalase precursor), mpa (escherichia coli, periplasmic murein-binding protein precursor), BglX (escherichia coli, periplasmic beta-glucosidase precursor), ArgT (escherichia coli, lysine-arginine-ornithine-binding periplasmic protein precursor), MalS (escherichia coli, alpha-amylase precursor), alpha-amylase precursor, beta-glucosidase precursor, and mixtures thereof, HisJ (e.coli, histidine-bound periplasmic protein precursor), XylF (e.coli, D-xylose-bound periplasmic protein precursor), FecB (e.coli, dicitrate-bound periplasmic protein precursor), OmpA (e.coli, outer membrane protein a precursor) and PhoA (e.coli, alkaline phosphatase precursor).

In a preferred embodiment, the signal sequence is selected from the group consisting of the e.coli signal peptides LamB (maltoporin precursor), MalE (maltose binding protein precursor), Bla (beta-lactamase), OppA (periplasmic oligopeptide binding protein), TreA (periplasmic trehalase precursor), mbppa (periplasmic murein peptide binding protein precursor), BglX (periplasmic beta-glucosidase precursor), ArgT (lysine-arginine-ornithine binding periplasmic protein precursor), MalS (alpha-amylase precursor), HisJ (histidine-binding periplasmic protein precursor), XylF (D-xylose-binding periplasmic protein precursor), FecB (dicitrate-binding periplasmic protein precursor), OmpA (outer membrane protein a precursor) and PhoA (alkaline phosphatase precursor). These signal sequences are particularly useful for heterologous expression in E.coli. More preferred are the E.coli signal peptides LamB (maltoporin precursor), MalE (maltose-binding protein precursor), Bla (. beta. -lactamase), TreA (periplasmic trehalase precursor), ArgT (lysine-arginine-ornithine binding periplasmic protein precursor), FecB (dicitrate-binding periplasmic protein precursor). Most particularly preferred are the E.coli signal sequences LamB (maltoporin precursor) and MalE (maltose-binding protein precursor). Where a dicistronic or polycistronic transcription unit is used, different or identical signal sequences may be operably linked to each cistron. Preferably, different signal sequences are used in this case. The signal sequence for use in the expression vector of the invention may be obtained from commercial sources or by chemical synthesis. For example, the signal sequence may be synthesized according to the solid phase phosphoramidite triester method as described by Beaucage & Caruthers (Tetrahedron letters.22: 1859-1862, 1981) using an automated synthesizer such as that described by Van Devanter et al (Nucleic Acids Res.12: 6159-6168, 1984). Oligonucleotides can be purified by native acrylamide gel electrophoresis or anion exchange HPLC as described by Pearson & Reanier (J.Chrom.255: 137-149, 1983).

A transcriptional unit of the invention will typically further comprise a translation initiation region upstream of the translation initiation site of said transcriptional unit, said translation initiation region consisting of the sequence AGGAGATATACAT (SEQ ID NO: 2), and the translation initiation region being operably linked to the nucleic acid sequence. The sequence AGGAGATATACAT (SEQ ID NO: 2) is generally located upstream of and directly adjacent to the translation start site (typically ATG, GTG or TTG) of the transcription unit.

Typically, the transcriptional unit further comprises a transcriptional termination region selected from the group consisting of rrnB, RNA I, T7Te, rrnB T1, trp a L126, trp a, tR2, T3Te, P14, tonB T, and trp a L153. Preferably, rrnB transcription termination sequences are used.

The heterologous nucleic acid sequence of the invention encodes an expression product that is heterologous to the host. In case the host is a prokaryotic species, such as E.coli, the nucleic acid sequence of interest is more preferably obtained from another class, such as gamma proteobacteria, such as from Burkholderia (Burkholderia sp.), especially from a different phylum, such as archaea, more preferably from a eukaryote, such as a mammal, especially a human. However, the heterologous nucleic acid sequence may be modified according to the "codon usage" of the host. The heterologous sequence of the invention is typically a gene of interest. The gene of interest preferably encodes a heterologous polypeptide, such as a structural, regulatory or therapeutic protein, or an N-or C-terminal fusion or other fusion of a structural, regulatory or therapeutic protein with another protein ("tag protein"), such as green fluorescent protein. The heterologous nucleic acid sequence may also encode a transcript that can be used in the form of RNA, such as antisense RNA.

The protein may be produced in the form of insoluble aggregates or solubilized proteins present in the cytosolic or periplasmic space of the host cell, and/or in the extracellular matrix. Preferably, the protein is produced as a solubilized protein present in the periplasmic space of the host cell and/or in the extracellular matrix. Examples of proteins include: hormones (such as growth hormone), growth factors (such as epidermal growth factor), analgesic substances (such as enkephalin), enzymes (such as chymotrypsin), antibodies, hormone receptors, and also proteins commonly used as visual markers (such as green fluorescent protein).

Other proteins of interest are growth factor receptors (such as FGFR, PDGFR, EFG, NGFR and VEGF) and their ligands. Other proteins are G-protein receptors, including the substance K receptors, angiotensin receptors, [ alpha ] -and [ beta ] -adrenoceptors, 5-hydroxytryptamine receptors, and PAF receptors (see e.g., Gilman, Ann. Rev. biochem.56, 625-649, 1987). Other proteins include ion channels (e.g., calcium, sodium, potassium channels), muscarinic receptors, acetylcholine receptors, GABA receptors, glutamate receptors, and dopamine receptors (see Harpold, U.S. Pat. Nos. 5401629 and 5436128). Other proteins of interest are adhesion proteins such as integrins, selectins (selecting), members of the immunoglobulin superfamily (see Springer, Nature346, 425-433 (1990); Osborn, Cell62, 3 (1990); Hynes, Cell69, 11 (1992)). Other proteins are Cytokines such as interleukins IL-1 to IL-3, tumor necrosis factors [ alpha ] and [ beta ], interferons [ alpha ], [ beta ] and [ gamma ], tumor growth factor beta (TGF-beta), Colony Stimulating Factor (CSF) and granulocyte monocyte colony stimulating factor (GM-CSF) (see Human Cytokines: Handbook for Basic & Clinical Research, "Human Cytokines: A Manual & Clinical Research, edited by Aggrawal et al, Blackwell Scientific, Boston, Mass., 1991). Other proteins of interest are extracellular or intracellular messengers, such as adenylate cyclase, guanylate cyclase, and phospholipase C. Drugs are also proteins of interest. The heterologous protein of interest may be of human, mammalian or prokaryotic origin. Other proteins are antigens such as glycoproteins and carbohydrates obtained from microbial pathogens, including viruses and bacteria, and from tumors. Other proteins are enzymes such as rennin, proteases, polymerases, dehydrogenases, nucleases, glucanases, oxidases, alpha-amylases, oxidoreductases, lipases, amidases, nitrile hydratases, esterases or nitrilases.

Preferably, the heterologous nucleic acid sequence of the invention encodes a polypeptide, more preferably an antibody, most preferably a Fab fragment. In particular, the nucleic acid sequence encodes a human or humanized antibody, more particularly a human Fab fragment. The human Fab fragment encoded by the nucleic acid sequence is preferably a human antibody fragment or a human antibody fragment grafted with at least one CDR derived from another mammalian species. In a particularly preferred embodiment, the human Fab fragment is a fully human HuCAL-Fab, obtained from an artificial consensus framework-based human antibody phage library, in which the CDRs are artificially randomized, as described by Knappik et al (2000, J.mol.biol., 296(1), 57-86).

In another more preferred optional embodiment, the optionally chimeric CDR-grafted human Fab fragment is a non-HuCAL-Fab as opposed to the foregoing HuCAL Fab definition, which in the case of the intact human Fab fragment preferably does not contain HuCAL consensus framework sequences, but whose non-CDR sequence portions are at least 70%, more preferably at least 85%, most preferably at least 95% identical in amino acid sequence to the corresponding variable and constant light and heavy chain germline coding sequences, and further and more preferably whose CDRs are directly derived from naturally occurring genomic sequences of lymphocytes including genomic affinity maturation events.

Fab fragments are preferably derived from IgG antibodies and do not contain cysteine residues that form the two interchain disulfide bonds between the two heavy chains of an intact immunoglobulin. In particular, the heavy and light chains of the antibody or preferably the Fab fragment are encoded by a dicistronic transcriptional unit, whereas each chain is operably linked to a prokaryotic signal sequence selected from the group consisting of carbohydrates, amino acids, vitamins and periplasmatic binding proteins for examples and the same translation initiation region upstream of the initiation site of the translation of the transcriptional unit. The translation initiation region preferably consists of the sequence AGGAGATATACAT (SEQ ID NO: 2).

In the present invention, the order and distance of the signal sequence and the heterologous nucleic acid sequence in the expression vector may be varied. In a preferred embodiment, the signal sequence is 5' to (upstream of) the nucleic acid sequence encoding, for example, a polypeptide of interest. The signal peptide sequence and the nucleic acid sequence encoding, for example, a polypeptide of interest may be separated by 0 to about 1000 amino acids. In a preferred embodiment, the signal peptide sequence and the nucleic acid sequence encoding e.g.the polypeptide of interest are directly adjacent to each other, i.e.separated by 0 amino acids.

Preferably, the promoter region and the operably linked transcription unit of the vector rhaBAD of the invention consist of the nucleotide sequence of SEQ ID NO: 3. its complementary sequence and its variant.

More preferably, the rhaBAD promoter region and the operably linked transcription unit of the vector of the invention consist of the amino acid sequence of SEQ ID NO: 4. its complementary sequence and its variant.

The invention also includes the use of the vectors of the invention for the regulated heterologous expression of nucleic acid sequences in prokaryotic hosts. Expression can be regulated by the amount of L-rhamnose available to the prokaryotic host. In general, the amount of L-rhamnose in the medium of the cultured prokaryotic host is from 0.01 to 100g/L, preferably from 0.1 to 10g/L, more preferably from 1 to 5 g/L.

Preferably, the heterologous nucleic acid sequence encodes a polypeptide, more preferably an antibody, most preferably a Fab fragment, while the heavy and light chains of the antibody or the Fab fragment are expressed in equal amounts, thereby producing a high concentration of functional antibody or Fab fragment. In particular, the heterologous nucleic acid sequence encodes a human or humanized antibody as described above, more preferably a human Fab antibody as described above, most preferably a human Fab fragment as described above.

To obtain a high concentration of functional antibody or Fab fragment, it is necessary to express equal amounts of heavy and light chains. When one of the two chains is overproduced compared to the other, non-reducible high molecular weight immunoreactive aggregates may or may not be produced, which is disadvantageous. Surprisingly, it has been found that high titers of functional antibodies can be obtained using the vectors of the invention, while producing only very small amounts of overproduced light or heavy chains or high molecular weight immunoreactive aggregates. Typically, less than 20%, preferably less than 10%, of the expressed amount of antibody or Fab fragment is expressed as an overproduced light or heavy chain or high molecular weight immunoreactive aggregate. The amount of overproduced light chain, heavy chain and high molecular weight immunoreactive aggregates can be determined by SDS-PAGE or Western blot analysis of extracts of host expressed antibodies or Fab fragments, such as lysozyme extracts of cultured host cells.

In another aspect, the invention provides an isolated and purified nucleic acid sequence expressed in a host, the sequence comprising the rhaDAB promoter region of the L-rhamnose operon operably linked to a transcriptional unit comprising

a) A nucleic acid sequence which is heterologous to the host,

b) a prokaryotic signal sequence selected from the group consisting of signal peptides of periplasmatic binding proteins for sugars, amino acids, vitamins and ions operably linked to said nucleic acid sequence, the expression of which is controlled by said promoter region.

The rhaBAD promoter is a preferred promoter region. More preferably, the isolated and purified nucleic acid sequence consists of SEQ ID NO: 1. the complementary sequences and variants thereof, in particular consisting of SEQ ID NO: 3. the isolated and purified nucleic acid sequence of the complement and variants thereof, most preferably consisting of SEQ ID NO: 4. the complementary sequence and variant thereof.

The isolated and purified nucleic acid sequences of the present invention can be isolated using standard PCR protocols and methods well known in the art. As is well known in the art, the isolated and purified DNA sequence may also contain one or more regulatory sequences, such as enhancers, which are commonly used in the expression of the product encoded by the nucleic acid sequence.

To select host cells that are successfully and stably transformed with the vectors of the invention or the isolated and purified nucleic acid sequences, a gene encoding a selectable marker (e.g., antibiotic-resistant) can be introduced into the host cell along with the nucleic acid sequence of interest. The gene encoding the selectable marker may be located on the vector, or on an isolated and purified nucleic acid sequence, or may optionally be co-introduced in an isolated form, e.g., on an isolated vector. Various selectable markers may be used, including markers that confer resistance to antibiotics (such as hygromycin, ampicillin, and tetracycline). The amount of antibiotic can be adjusted as needed to create selective conditions. Typically, a selectable marker is used. A reporter gene (e.g., a fluorescent protein) can also be introduced into the host cell along with the nucleic acid sequence of interest to determine transformation efficiency.

Another aspect of the invention provides a prokaryotic host transformed with a vector of the invention. In a particular embodiment of the invention, the prokaryotic host is transformed with plasmid pBW22-Fab-H or pAKL14, preferably plasmid pAKL14, which contains two different coding regions in the dicistronic expression cassette for expression of a secreted heterodimeric protein (e.g., Fab) in the host cell. Preferably, such heterodimeric protein is a Fab. In another embodiment of the invention, a prokaryotic host is transformed with an isolated and purified nucleic acid sequence of the invention.

A wide range of prokaryotic host cells can be used to express the heterologous nucleic acid sequences of the invention. These hosts may include gram-negative cell strains, such as E.coli and Pseudomonas (Pseudomonas); or gram-positive cell strains, such as Bacillus (Bacillus) and Streptomyces (Streptomyces). Preferably, the host cell is a gram-negative cell, more preferably an E.coli cell. Coli which can be used are, for example, the strains TG1, W3110, DH1, XL1-Blue and Origami, which are commercially available or are obtainable by DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). Most preferably, W3110 is used. The host cell may or may not metabolize L-rhamnose. Host cells that are normally capable of uptake and metabolism of L-rhamnose, such as E.coli, may be modified to lack one or more of the functions associated with the uptake and/or metabolism of L-rhamnose. Defects in one or more functions associated with the uptake and/or metabolism of L-rhamnose can be achieved, for example, by inhibiting or blocking a gene which codes for a protein which is involved in the uptake and/or metabolism of L-rhamnose, for example the rhaB gene which codes for L-rhamnose kinase. This can be achieved using known techniques such as transposon-supported mutagenesis or knockout mutation. Usually, the prokaryotic host corresponds to the selected signal sequence, as in the case of using signal sequences of E.coli, the host cell is usually the same member of the family of Enterobacteriaceae, more preferably the host is an E.coli strain.

Also provided by the present invention is a method of producing a polypeptide in a host cell, the method comprising the steps of:

a) constructing a vector;

b) transforming a prokaryotic host with the vector;

c) under appropriate conditions, allowing the polypeptide to be expressed in a cell culture system;

d) recovering the polypeptide from the cell culture system.

The vectors used, as well as their construction and transformation of prokaryotic hosts, are as defined above, and the heterologous nucleic acid sequences contained in the vectors encode polypeptides. Preferably, the polypeptide produced is an antibody, most preferably a Fab fragment, and the heavy and light chains of the antibody or Fab fragment are expressed in equal amounts in a cell culture system, thereby producing a high concentration of functional antibody or Fab fragment.

For cell culture systems, continuous or discontinuous cultures such as batch cultures or fed-batch cultures can be used in culture tubes, shake flasks or bacterial fermentors. The host cells are usually cultured in conventional media known in the art, for example complex media such as "nutrient yeast broth" or in glycerol-containing media as described by Kortz et al (1995, J.Biotechnol.39, 59-65) or in mineral salt media as described by Kulla et al (1983, Arch.Microbiol, 135, 1). Preferred media for carrying out the expression of the polypeptide are glycerol-containing media, more preferably the media described by Kortz et al (1995, J.Biotechnol.39, 59-65).

The medium may be appropriately changed, for example, by further adding other components such as buffer, salt, vitamins or amino acids. Different media or combinations of media can also be used during the culturing of the cells. Preferably, the medium used as basal medium should not contain L-rhamnose in order to achieve a tight regulation of the L-rhamnose promoter region. In generalTo achieve a suitable OD in the culture medium₆₀₀L-rhamnose was added after (depending on the culture system). In general, the amount of L-rhamnose in the medium of the cultured prokaryotic host is from 0.01 to 100g/L, preferably from 0.1 to 10g/L, more preferably from 1 to 5 g/L. For batch cultures, the typical OD600 is usually 0.4 or higher. A suitable pH range is, for example, 6-8, preferably 7-7.5, and a suitable cultivation temperature is 10-40 ℃, preferably 20-37 ℃. In general, the cells are cultured for as long a time as possible until the maximum amount of the expression product is accumulated, and the time is preferably 1 hour to 20 days, more preferably 5 hours to 3 days. The amount of expression product depends on the culture system used. In shake flask culture, a host transformed with a vector of the invention will typically produce 0.5g of expression product per liter of medium. In a fermenter culture in batch and/or fed-batch mode, it is generally possible to produce more than 0.5g, preferably more than 1g, more preferably more than 1.3g, of expression product per liter of fermentation broth.

Following expression by the host cell, the expression product, e.g., the polypeptide of interest, can be recovered from the host cell culture. When the polypeptide of interest is an immunoglobulin chain, the heavy and light chains are each typically expressed in the host cell and secreted into the periplasm of the cell. The signal peptide encoded by the signal sequence in the expression vector is then processed from the immunoglobulin chain. The mature heavy and light chains are then assembled into complete antibodies or Fab fragments. To obtain the maximum yield of expression product, the cells are usually harvested at the end of the culture and lysed, for example by lysozyme treatment, sonication or French Press. Thus, the polypeptide is usually first obtained as a crude lysate of the host cell. They can then be purified using standard protein purification methods known in the art, which may include differential precipitation, molecular sieve chromatography, ion exchange chromatography, isoelectric focusing, gel electrophoresis, affinity, and immunoaffinity chromatography. These well known and conventionally practiced methods are described in, for example, ausable et al (supra) and Wu et al (ed) Academic Press inc, n.y.; immunochemical Methods in cell And Molecular Biology (Immunochemical Methods in cell And Molecular Biology) are described. For example, to purify recombinantly produced immunoglobulins or Fab fragments, they can be purified by passing them through a column containing a resin to which the target molecule to which the expressed immunoglobulin specifically binds is bound using immunoaffinity chromatography.

Another aspect of the invention is a vector expressible in a host operably linked to a promoter region of a transcriptional unit comprising:

a) a nucleic acid sequence which is heterologous to the host,

b) a translation initiation region upstream of the translation initiation site of said transcriptional unit consisting of the sequence AGGAGATATACAT (SEQ ID NO: 2) the structure of the utility model is that the material,

the translation initiation region is operably linked to the nucleic acid sequence, and expression of the nucleic acid sequence is controlled by the promoter region. The promoter region may be an inducible or non-inducible promoter region. Usually, an inducible promoter region of the catabolic operon is used. Inducible promoter regions of promoter systems in which the catabolic operon is negatively regulated can be used, such as the lactose [ lac ] (Yanisch-Perron et al, 1985, Gene33, 103-109), and tryptophan [ trp ] (Goeddel et al, 1980, Nature (London) 287, 411-416) promoters, and hybrid promoters [ tac and trc ] (Brosius, 1984, Gene 27: 161-172; Amann and Brosius, 1985, Gene40, 183-190) derived from both promoters, as well as positively regulated promoter systems, such as the ara B promoter induced by arabinose (WO8604356), the rhamnose promoter rhaSB (WO03068956), or the "rhaBAD promoter region of the rhamnose operon" as described herein. It is preferred to use positively regulated catabolic promoters, more preferably the "rhaBAD promoter region of the L-rhamnose operon" of the present invention. Functional equivalents of these promoters, which may be obtained from various prokaryotes, may also be used. Functional equivalents in the case of positively regulated catabolic operon equivalents are equivalents which exhibit increased expression activity in the presence of an inducer compared to the activity they possess in the absence of an inducer. The expression activity in the presence of an inducer is generally 2-fold, preferably at least 5-fold, more preferably at least 10-fold higher than the expression activity in the absence of an inducer.

Typically, the vector also contains a signal sequence operably linked to the nucleic acid sequence. The signal sequence may be prokaryotic or eukaryotic. Preferably, prokaryotic signal sequences are used. As mentioned above, the prokaryotic signal sequence is preferably selected from the signal peptides of periplasmatic binding proteins for sugars, amino acids, vitamins and ions, or from other prokaryotic signal sequences known to the person skilled in the art. More preferably, the prokaryotic signal sequence is selected from the group consisting of signal peptides of periplasmatic binding proteins for sugars, amino acids, vitamins and ions as described above. Typically, as described above, the nucleic acid sequence encodes a polypeptide, preferably an antibody, more preferably a Fab fragment.

In a particular embodiment, in case the nucleic acid sequence encodes an antibody, preferably the Fab fragment, the heavy and light chains of the antibody, preferably the Fab fragment is encoded by a dicistronic transcriptional unit, each chain being operably linked to a signal sequence and a translation initiation region consisting of the sequence AGGAGATATACAT (SEQ ID NO: 2).

In a further aspect the present invention provides a method for producing a polypeptide in a host cell, the method comprising the steps of:

a) constructing a vector;

b) transforming a prokaryotic host with the vector;

c) under appropriate conditions, allowing the polypeptide to be expressed in a cell culture system;

d) recovering the polypeptide from the cell culture system.

The vectors and hosts used are as defined above. The construction of the vector, the transformation of the prokaryotic host and the cell culture may be carried out according to the above description, the heterologous nucleic acid sequence comprised by the vector encoding the polypeptide. In case the polypeptide produced is a Fab fragment, the heavy and light chains of the Fab fragment are expressed in the cell culture system in the same amount.

The invention also relates to methods and means for the intracellular heterologous expression of nucleic acids encoding e.g. polypeptides in prokaryotic hosts. In particular, the present invention relates to a vector for the intracellular expression of a heterologous polypeptide in a prokaryotic host, wherein the vector is expressed in the prokaryotic host and comprises the rhaBAD promoter region of the L-rhamnose operon operably linked to a transcriptional unit comprising a nucleic acid sequence which is heterologous to said host. Since in the vector embodiments of the invention the nucleic acid sequence is not linked to a prokaryotic signal sequence, upon transformation of a prokaryotic host cell with the vector and expression of the polypeptide encoded by the heterologous nucleic acid, the polypeptide will not be transferred from the cytoplasm to a non-cytoplasmic location. Instead, the polypeptide will be expressed in the cytoplasm in either the endosomal or soluble form. Thus, following expression, the polypeptide may be isolated and purified from the cell, particularly from cell extracts, using well-known methods. The invention also provides the use of said vector for the regulated intracellular expression of a heterologous nucleic acid sequence in a prokaryotic host; a prokaryotic host or prokaryotic host cell transformed with the vector; a method for the intracellular production of a heterologous polypeptide in a prokaryotic host using said vector; and a vector for the intracellular production of a polypeptide, wherein the vector comprises a promoter region, a heterologous nucleic acid sequence encoding a heterologous polypeptide, and a translation initiation region consisting of the sequence aggagatatatacat.

In a preferred embodiment, where a vector is used for intracellular expression, the rhaBAD promoter is represented by SEQ ID NO: 1, its complement and variants thereof. Preferably, said rhaBAD promoter region and said operably linked transcriptional unit are encoded by SEQ ID NO: 3 or SEQ ID NO: 4. its complementary sequence or its variant sequence. According to the invention, the vector for intracellular expression may contain a dicistronic transcription unit. In another preferred embodiment of the invention, the transcription unit of the vector further comprises a translation initiation region upstream of the translation initiation site of said transcription unit, and said translation initiation region consists of the sequence AGGAGATATACAT (SEQ ID NO: 2). In another preferred embodiment, the vector for intracellular expression contains a transcription termination region, such as the rrnB transcription termination sequence. According to the invention, the heterologous nucleic acid sequence may encode a polypeptide, such as an antibody, an antibody fragment, or the like.

It will be understood by those skilled in the art that modifications and variations may be made to the invention described herein beyond those specifically described herein. It is to be understood that the invention includes all such modifications and alterations without departing from the spirit and essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The disclosure is therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein. Each reference is cited throughout the specification and is incorporated herein by reference.

The foregoing description will be more fully understood in conjunction with the following examples. However, these examples are exemplary methods of practicing the invention and they do not limit the scope of the invention.

Examples

Example 1

Construction of expression plasmids with positively regulated promoters

The genome of E.coli W3110 was searched for positively regulated operons. Positively regulated catabolic promoters were identified and analyzed for their use in expression plasmids based on genomic data available from the KEGG database (Kyoto Encyclopedia of Genes and Genomes, http:// www.genome. adjp/KEGG 2. html). The promoter should be tightly regulated and induced in an inexpensive and non-toxic manner and therefore be an industrially useful compound. Promoters were selected for the following different positively regulated catabolic operons:

the prp promoter (propionate-induced)

-the gutA promoter (glucitol induced)

The melAB2 promoter (melibiose-induced)

The precise DNA fragment containing the promoter element is selected based on the information available on the corresponding regulatory binding site. Chromosomal DNA of E.coli was isolated using the method described by Pitcher et al (1989, Letters in Applied Microbiology8, 151-156). A promoter fragment was amplified from chromosomal DNA of strain W3110 by PCR using the following primers. Restriction sites for ClaI and AflII are underlined. The sequences of these fragments are shown below:

Pprp Pprp-5 5′aaa atc gat aaa tga aac gca tat ttg3’

Pprp-3 5′aaa ctt aag ttg ttatca acttgttat3’

AAAATCGATAACTGAAACGCATATTTGCGGATTAGTTCATGACTTTATCTCTAACAAA

TTGAAATTAAACATTTAATTTTATTAAGGCAATTGTGGCACACCCCTTGCTTTGTCTTT

ATCAACGCAAATAACAAGTTGATAACAACTTAAGTTT

PgutA PgutA-5 5’aaa atc gat gca tca cgc ccc gca caa3’

PgutA-3 5’aaa ctt aag tca gga ttt att gtt tta3’

AAAATCGATGCATCACGCCCCGCACAAGGAAGCGGTAGTCACTGCCCGATACGGAC

TTTACATAACTCAACTCATTCCCCTCGCTATCCTTTTATTCAAACTTTCAAATTAAAATA

TTTATCTTTCATTTTGCGATCAAAATAACACTTTTAAATCTTTCAATCTGATTAGATTAG

GTTGCCGTTTGGTAATAAAACAATAAATCCTGACTTAAGTTT

PmeIAB2 PmeIAB-5-1 5’aaa atc gat gac tgc gag tgg gag cac3’

PmeIAB-3 5’aaa ctt aag ggc ttg ctt gaa taa ctt3’

MeIR CRP

AAAATCGATATT

GCGTTTTCTGCA

GATTCGCCTGCCATGATGAAGTTATTCAAGCAAGCCCTTAAGTTT

+1

(binding sites for CRP2 are marked in light grey while binding sites for MelR are marked in black)

The fragments were separated by agarose gel electrophoresis and isolated using the gel extraction kit QiaexII from Qiagen (Hilden, Germany). The isolated fragments were cleaved with ClaI and AflII and ligated into CalI/AflII-cleaved pBW22(Wilms et al, 2001, Biotechnology and Bioengineering, 73(2), 95-103). The resulting plasmids containing the prp promoter (pBLL5), the gutA promoter (pBLL6) and the melAB2 promoter (pBLL7) were identical except for the linked promoter region. The sequence of the inserted promoter was determined by sequencing (Microsynth GmbH, Balgach, Switzerland).

Example 2

Construction of Fab fragment expression plasmid

The ability of different positively regulated expression systems to produce Fab-H antibody fragments was analyzed as an alternative to the IPTG-inducible lac promoter (plasmid pMx9-HuCAL-Fab-H, Knappik et al, 1985, Gene33, 103-119). Fab-H fragments were amplified from plasmid pMx9-HuCAL-Fab-H using PCR with primers Fab-5 (5'-aaa cat atg aaa aag aca gct atc-3') and Fab-3 (5'-aaa aag ctt tta tca gct ttt cgg ttc-3'). The PCR fragment was cut with NdeI and HindIII and inserted into NdeI/HindIII cut pBW22(Volff et al, 1996, mol. Microbiol.22, 1037-1047) to generate plasmid pBW22-Fab-H (FIG. 1) containing the rhamnose inducible rhaBAD promoter (SEQ ID NO: 1). The same PCR fragment was inserted into a different expression plasmid with an inducible promoter. The resulting (putative) Fab-H containing expression plasmids were pBLL13 containing the prp promoter, pBLL14 containing the gutA promoter and pBLL15 containing the melAB2 promoter (FIG. 2). The sequence of the Fab-H insert in plasmid pBW22-Fab-H was determined by sequencing.

Example 3

Expression of Fab fragments

The strain W3110 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) was transformed with different expression vectors. Plasmids were isolated from clones generated from different transformations and checked by restriction analysis. All plasmids except plasmid pBLL14 had the expected restriction pattern. The re-isolated plasmid pBLL14 showed an altered size and restriction pattern, which was suggested to be the result of a recombination event. Therefore, strain W3110(pBLL 14) was not tested in the following experiments. The remaining strains were tested for their ability to secrete actively folded Fab-H antibody fragments. This yield test was performed as described in example 4. The following inducers were added at a concentration of 0.2%:

pBW22-Fab-H L (+) -rhamnose monohydrate

pBLL13 sodium propionate

pBLL 15D (+) -melibiose monohydrate

D (+) -raffinose monohydrate

D (+) -galactose

The results of the dot blot assay are shown in figure 3.

The rhamnose and melibiose induced strain W3110(pBW22-Fab-H) and W3110(pBLL15) showed the expected dot blot results: the signal increased over time with little background activity. The actively folded antibody fragment fraction was quantified by ELISA. The results are summarized in Table 1 below.

Table 1: ELISA results of W3110 derivatives with different expression plasmids. The time after induction is shown. Uninduced cultures after 22 hours or 25 hours were tested as uninduced controls and the results from strain W3110(pMx9-HuCAL-Fab-H) and TGlF' - (pMx9-HuCAL-Fab-H) were used as controls. Concentration of Fab-H in mg/100OD₆₀₀the/L meter (nd means not measured).

All strains being present or absentGood growth without any growth inhibition, OD in the case of the corresponding inducer₆₀₀To between 4 and 6. Expression plasmids pBW22-Fab-H (containing SEQ ID NO: 3) and pBLL15 produced the highest antibody fragment titers after overnight induction. The melibiose-induced strain W3110(pBLL15) showed a delayed increase in the formation of active antibody fragments compared to the rhamnose (pBW22-Fab-H) induced system.

The rhamnose-inducible strain W3110(pBW22-Fab-H) was tested in a respiratory activity monitoring system (RAMOS, ACBiotec, Julich, Germany), a novel measurement system for the real-time determination of respiratory activity in shake flasks. Compared to normal shake flask experiments, the antibody titer (measured by ELISA) doubled (703.64 mg/L/100OD 23 hours after induction)₆₀₀). Optimal growth using RAMOS equipment favors the production of active antibody fragments.

Example 4

Melibiose Induction in Shake flasks

Coli W3110 harboring plasmid pBLL15 was tested for its ability to produce actively folded Fab-H antibody fragments. The overnight culture [ in NYB medium (10g/l tryptone, 5g/l yeast extract, 5g/l sodium chloride) supplemented with 100. mu.g/ml ampicillin, 37 ℃ C. (Amersham pharmacia Biotechnol., 39, 59-65) was diluted (1: 50) in 20ml fresh glycerol medium (as described by Kortz et al, 1995, J.Biotechnol., 39, 59-65, where the vitamin solution was used according to Kulla et al, 1983, Arch. Microbiol, 135, 1)]And incubated at 30 ℃. When the culture reached an OD of about 0.4₆₀₀Melibiose (0.2%) was added at this time. Samples (1ml) were taken at different time intervals, centrifuged and the pellet was stored at-20 ℃. Frozen cells were lysed according to the above lysozyme treatment and supernatants were analyzed in dot blot and ELISA assays. 504.28mg/L/100OD was obtained₆₀₀A functional Fab-H antibody fragment of (1).

Example 5

Appearance of high molecular weight aggregates

To find out whether high molecular weight aggregates were produced, extracts of strain W3110(pBLL15) showing the highest antibody titer (Table 1) were subjected to Western blotting using anti-human Fab-H + AP conjugate. Incubations were performed as described in example 4. Samples were taken at 9, 12 and 23 hours after melibiose induction. Lower concentrations of high molecular weight aggregates correspond to higher titers of functional antibody fragments. The choice of expression system appears to influence the way antibody formation: functionality is also in the form of aggregates.

Example 6

Effect of Signal peptide

The genomic database of E.coli was used to find useful signal peptides that could be used with the Fab-H fragments VL3-CL and VH-CH. Signal peptides derived from periplasmic binding proteins of sugars, amino acids, vitamins and ions were selected. These periplasmic proteins represent a group of relatively identical species (homogeneous) that have been studied more extensively than other periplasmic proteins. Since they are usually abundant, their signal sequence has to ensure efficient transport across the inner membrane and into the cytoplasm. The sequence peptide and dissociation site probabilities for all possible signal peptide Fab combinations were examined using the SignalP Web Server (http:// www.cbs.dtu.dk/services/SignalP-2.0/# Transmission), as shown in Table 2 below.

The following 6 combinations were selected:

-LamB-VL 3-CL (maltoporin precursor)

-MalE-VH-CH (maltose binding protein precursor)

-Bla-VL 3-CL (beta-lactamase)

-TreA-VH-CH (periplasmic trehalase precursor)

ArgT-VL3-CL (lysine-arginine-ornithine binding periplasmic protein precursor)

-FecB-VH-CH (ferric (III) dicitrate binding periplasmic protein precursor)

Gene fusions to generate fusions of Signal Peptide (SP) with VL3-CL and VH-CH were performed using overlapping PCR primers and are shown in amplification Table 3 below.

Fusion of the signal peptide sequence with the VL3-CL and VH-CH sequences was carried out as described elsewhere ((Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K., and Pease, L.R (1989) Engineering hybrid genes with out the use of restriction enzymes: Gene splicing by overlap extension.: engineered hybrid genes without restriction enzymes: Gene splicing by Gene splicing [ Gene77, 61-68 ]. cleavage of the SP-VL3-CL Gene with restriction enzymes NdeI and PstI, integration into NdeI/tI cleaved pBW22, and ligation into pBLL 7. plasmid cleaved with PstI and HindIII, plasmid cleaved with PstI/HindIII ligated SP-CH 5 and VH-CH 5 containing VH-VL-B Gene, which was not possible to obtain expression of the Fab-VL Gene alone, and VH-VL-CL containing the Gene was detected because the expression of the Gene was only the rhamnose-VL-CL and VH-VL-CL containing the inducible plasmid VL3-CL/male-VH-CH expression plasmid (pAKL 14). The lamB-VL3-CL/malE-VH-CH genes isolated as AflII/HindIII fragments from pAKL15 (example 7) were ligated to AflII/HindIII cut pBLL7 to obtain pAKL 15E. FIGS. 4 and 8 illustrate the lamB-VL3-CL/malE-VH-CH expression plasmids pAKL14 and pAKL 15E.

Example 7

Effect of translation initiation region on Fab expression

The Fab-H genes of plasmid pAKL14 (containing SEQ ID NO: 4) and plasmid pAKL15E contained the 5' end of the same DNA sequence of the start codon (translation initiation region), whereas in the original plasmid pMx9-HcCAL-Fab-H the translation initiation regions of the two Fab-H genes were different. A comparison of the sequences of the translation initiation regions of plasmids pMx9-HuCAL-Fab-H and pAKL14/pAKL15E is shown in Table 4 below:

pMx9-HuCAL-Fab-H ompA-VL3-CL gagggcaaaaa atg

phoA-VH-CH aggagaaaataaa atg

pAKL14/pAKL15E lamB-VL3-CL aggagatatacat atg

malE-VH-CH aggagatatacat atg

the yield of W3110(pAKL14) was tested in shake flasks as described in example 4. The strain grew well in the presence and absence of L-rhamnose. This means that the production of Fab-H does not affect the viability of the cells. As shown in fig. 5, the dot blot results appear to be perfect.

To analyze the presence of non-inducible high molecular weight aggregates, western blots were performed (fig. 6). Although high molecular weight aggregates appeared 5 hours after induction, their amount increased only slightly after 23 hours. Uninduced cultures showed high molecular weight bands, which may be weak non-specificThe production of an anisotropic background. The corresponding ELISA values are shown in Table 5 below (uninduced and rhamnose-induced concentration of Fab-H in lysozyme extract of strain W3110(pAKL14) (mg/L/100 OD)₆₀₀))。

The new signal peptide construct (with modified translation initiation signal) again brought the antibody titer from 328.62mg/L/100OD₆₀₀(containing the MOR gene construct from pMx9-HuCAL-Fab-H plasmid pBW22-Fab-H) improved to 596.14mg/L/100OD₆₀₀(plasmids pAKL14) and 878.86mg/L/100OD₆₀₀(plasmid pAKL 15E). Sequencing of the lamB-VL3-CL and malE-VH-CH genes in pAKL14 revealed three base exchanges, probably due to the construction of the fusion gene by two consecutive PCR reactions. Base exchanges result in the following amino acid changes (wrong amino acids are highlighted):

VL3-CL(pAKL14)-pI＝4.85

MMITLRKLPLAVAVAAGVMSAQAMADIELTQPPSVSVAPGQTARISCSGNALGDK

YASWYQQNPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISGTQAEDEADYY

CQSYDSPQVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPG

AVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT

HEGSTVEKTVAPTEA

VH-CH(pAKL14)-pI＝9.52

MKIKTGARILALSALTTMMFSASALAQVQLKESGPALVKPTQTLTLTCTFSGFSLST

SGVGVGWIRQPPGKALEWLALIDWDDDKYYSTSLKTRLTISKDTSKNQVVLTMTN

MDPVDTATYYCARYPVTQRSYMDVWGQGTLVTVSSASTKGPSVLPLAPSSKSTS

GGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKKVEPKS

the light chain of Fab-H has two errors (D50N and K63N) and the heavy chain has one amino acid exchange (F156L). To restore the original Fab-H sequence, the two fragments of plasmid pAKL14 (138bp SexAI/BamHI and 310bp BssHII/HindIII fragment) were exchanged against the homologous fragment of plasmid pBW22-Fab-H (with unaltered Fab-H gene sequence). The resulting plasmid pAKL15 carries the correct Fab-H sequence. The exchange of three amino acids did not have a significant effect on the overall Fab-H characteristics, since the pI was not changed. Thus, the ability of strain W3110(pAKL15) to produce functional Fab-H antibody fragments was considered to be similar to the ability of strain W3110(pAKL14) to produce, and was not analyzed.

The productivity of Fab-H antibody fragments can be increased by using different optimisation strategies. Table 6 below summarizes these improvements:

bacterial strains	Improvements in or relating to	Concentration of functional Fab-H antibody (mg/L/100 OD)600)	Increased activity
				TG1F’-(pMx9-HuCAL-Fab-H)	MOR strain	84.56
W3110(pMx9-HuCAL-Fab-H)	Background of the Strain	140.45	1.7
				W3110(pBW22-Fab-H)	Expression system (rhamnose)	328.62	3.9
W3110(pBLL15)	Expression system (melibiose)	504.28	6
				W3110(pAKL14)	Signal peptide translation (rhamnose)	596.14	7
W3110(pAKL15E)	Signal peptide translation (melibiose)	878.86	10.4

Strains producing high Fab-H antibody titers were analyzed by SDS-PAGE (FIG. 7). The highest functional Fab-H concentration was measured in strains producing balanced amounts of light and heavy chains (lanes 4 and 5). Rhamnose inducible strains with Fab-H fragments, such as W3110(pBW22-Fab-H) (lane 3), strongly overproduce the light chain.

Example 8

Induction of melibiose in Shake flasks

Coli W3110 (FIG. 8) carrying plasmid pAKL15E was tested for its ability to produce actively folded Fab-H antibody fragments. The overnight culture [ in NYB medium (10g/l tryptone, 5g/l yeast extract, 5g/l sodium chloride) supplemented with 100. mu.g/ml ampicillin, 37 ℃ C. (Amersham pharmacia Biotechnol., 39, 59-65) was diluted (1: 50) in 20ml fresh glycerol medium (as described by Kortz et al, 1995, J.Biotechnol., 39, 59-65, where the vitamin solution was used according to Kulla et al, 1983, Arch. Microbiol, 135, 1)]And incubated at 30 ℃. When the culture reached an OD of about 0.4₆₀₀Melibiose (0.2%) was added at this time. Samples (1ml) were taken at different time intervals, centrifuged and the pellet was stored at-20 ℃. Frozen cells were lysed according to the above lysozyme treatment and supernatants were analyzed in SDS-PAGE and ELISA assays. The melibiose inducible strain carrying the Fab-H gene with altered signal peptide (lamB-VL3-CL/malE-VH-CH) showed the highest Fab-H antibody titers (Table 6). The light and heavy chains of Fab-H were produced in equal amounts (FIG. 9).

Example 9

Intracellular production of antibody fragments

Origami host strains provide mutations in both the thioredoxin reductase (trxB) and glutathione reductase (gor) genes, enhance disulfide bond formation, and allow protein folding in the bacterial cytoplasm. To construct VL3-CL and VH-CH genes without signal peptide regions, the following primers were used:

5’-VL5’-aaa cat atggat atc gaa ctg acc cag-3' (NdeI restriction site)

3’-CL5’-aaa ctg cagtta tca ggc ctc agt cgg-3' (PstI restriction site)

5’-VH5’-aaa ctg caggag ata tac ata tgc agg tgc aat tga a-3' (PstI restriction site)

3 ' -CH5 ' -aaa aag ctt tta tca gct ttt cgg ttc-3 ' (HindIII restriction site)

The corresponding VL3-CL and VH-CH genes were amplified and checked by restriction analysis. The NdeI/PstI cut VL3-CH fragment was integrated into NdeI/PstI cut plasmid pBW 22. The resulting plasmid was cut with PstI and HindIII and ligated to the PstI/HindIII cut VH-CH fragment to give plasmid pJKL 6. This plasmid was transformed into Origami strain and W3110 strain, with W3110 strain as a reference. The yields of strain W3110(pJKL6) and Origami (pJKL6) were tested in shake flasks as described in example 4.

To analyze the presence of functional antibody fragments and non-inducible high molecular weight aggregates, western blots were performed. Both strains hardly produce any functional antibody fragments. With the passage of induction time (W3110), the W3110 strain accumulated high molecular weight aggregates, whereas the Origami strain did not produce any antibody fragments. The corresponding ELISA values (Fab-H concentrations (mg/L/100OD 6) in lysozyme extracts of the uninduced and rhamnose-induced Origami (pJKL6) and W3110(pJKL6) strains are given in Table 7 below₆₀₀))。

Example 10

Rhamnose Induction of Single chain antibodies (scFv, S1) in Shake flasks

The scFv gene was isolated by PCR using primers 5-S (5'-aaa cat atg aaa tac cta ttg cct acg gc-3') and 3-S1 (5'-aaa aag ctt act acg agg aga cgg-3'). The corresponding S1 protein contains the PelB signal sequence, which is responsible for the transport of the protein into the periplasm of E.coli. The PCR fragment was cut with NdeI and HindIII and inserted into NdeI/HindIII cut pBW22 to generate plasmid pBW22-pelB1-S1 containing the rhamnose inducible rhaBAD promoter (FIG. 10). The S1 insert sequence of plasmid pBW22-pelB-S1 was determined by sequencing. Strain W3110 (DSM5911, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) was transformed with plasmid pBW 22-pelB-S1. Plasmids were isolated from different clones and verified by restriction analysis. Coli W3110(pBW22-pelB-S1) was tested for its ability to produce soluble S1. The overnight culture [ in NYB medium (10g/l tryptone, 5g/l yeast extract, 5g/l sodium chloride) supplemented with 100. mu.g/ml ampicillin, 37 ℃ C. (Amersham pharmacia Biotechnol., 39, 59-65) was diluted (1: 50) in 20ml fresh glycerol medium (as described by Kortz et al, 1995, J.Biotechnol., 39, 59-65, where the vitamin solution was used according to Kulla et al, 1983, Arch. Microbiol, 135, 1)]And incubated at 30 ℃. When the culture reached an OD of about 0.4₆₀₀Rhamnose (0.2%) was added at the time. Samples (1ml) were taken at different time intervals, centrifuged and the pellet was stored at-20 ℃. Frozen cells were lysed according to the above lysozyme treatment and the supernatant and insoluble protein pellet were analyzed by SDS-PAGE (FIG. 11) and Bioanalyzer. Most of the S1 protein (mg/L/100 OD)₆₀₀) Produced as a soluble protein fraction.

Example 11

Construction of a broad host range rhamnose expression plasmid for Pseudomonas and related bacteria

The following cloning experiments were carried out in E.coli JM 109. The broad-host-range cloning vector pBBR1MCS-2(NCBI accession No. U23751) was cut with AgeI/NsiI. lacZ α gene was deleted and replaced with oligo nucleotides 3802 (5'-tgt taa ctg cag gat cca agc tta-3') and 3803 (5'-ccg gta agc ttg gat cctgca gtt aac atg ca-3') to give plasmid pJOE4776.1. The rhaRSP fragment was provided using plasmid pJKS408 (unpublished) containing the genomic rhaRS fragment (2kb) of Escherichia coli JM 109. Plasmid pJKS408 was cut with BamHI/HindIII, ligated to the eGFP fragment (0.7kb) of BamHI/HindIII cut plasmid pTST101 [ Stumpp, T., Wilms, B., Altenbuchner, J. (2000): ein neues, L-Rhamnose-indzierbares expression system fur Escherichia coli, Biospecrum6, 33-36 ]. A rhaRSPmalE-eGFP fragment (4kb) was isolated from the resulting plasmid pJOE4030.2 using NsiI/HindIII and integrated into NsiI/HindIII-cut pJOE4776.1. Plasmid pJOE4776.1 (FIG. 12) contains the rhaBAD promoter region in combination with the genes for the regulatory proteins RhaS and RhaR of the rhamnose operon of E.coli in a broad-host-range plasmid backbone.

Example 12

Rhamnose Induction of nitrilases in Shake flasks

The nitrilase gene was cut with NdeI and BamHI from plasmid pDC12(Kiziak et al, 2005) and inserted into NdeI/BamHI cut pJOE4782.1 to give plasmid pAKLP2 containing the L-rhamnose inducible rhaBAD promoter (FIG. 13). As an intermediate step, E.coli XL1-Blue strain was transformed with plasmid pAKLP 2. Plasmids were isolated from different clones and verified by restriction analysis. Pseudomonas putida KT-2440(Pseudomonas putida strain KT-2440, D SM6125, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) was transformed with the isolated plasmid pALKP2 obtained from E.coli XL1Blue (pAKLP 2). Pseudomonas putida KT-2440 was tested for its ability to produce nitrilase. At 20mThe overnight cultures were diluted [ in NYB medium (10g/l tryptone, 5g/l yeast extract, 5g/l sodium chloride) supplemented with 50. mu.g/ml kanamycin, 30 ℃ C. (as described by Kortz et al, 1995, J.Biotechnol.39, 59-65, with the exception of vitamin solutions (as described by Kulla et al, 1983, Arch. Microbiol., 135, 1)) ]]Make OD₆₀₀About 0.1 and incubated at 30 ℃. When the culture reached an OD of about 0.25₆₀₀L-rhamnose (1.0%) was added at that time. Samples (1ml) were taken at different time intervals, centrifuged and the pellet was stored at-20 ℃. The pellet was resuspended in Tris/HCl buffer (50mM, pH8.0) and the cell suspension was analyzed by SDS-PAGE (FIG. 14).

Example 13

L-rhamnose Induction of fragment antibodies (FabM) in Shake flasks

The Fab-M gene was cleaved from plasmid pBW22-FabM with NdeI and BamHI and inserted into NdeI/BamHI-cleaved pJOE4782.1 to generate plasmid pAKLP1 containing the L-rhamnose inducible rhaBAD promoter (FIG. 15). As an intermediate step, E.coli XL1-Blue strain was transformed with plasmid pAKLP 1. Plasmids were isolated from different clones and verified by restriction analysis. Pseudomonas putida KT-2440(Pseudomonas putida strain KT-2440, DSM6125, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) was transformed with the isolated plasmid pALKP1 obtained from E.coli XL1Blue (pAKLP 1). Pseudomonas putida KT-2440(pAKLP1) was tested for its ability to produce Fab-M. The overnight cultures [ in NYB medium (10g/l tryptone, 5g/l yeast extract, 5g/l sodium chloride) supplemented with 50. mu.g/ml kanamycin, 30 ℃ C., were diluted in 20ml fresh glycerol medium (as described by Kortz et al, 1995, J.Biotechnol.39, 59-65, with the exception of vitamin solutions (as described by Kulla et al, 1983, Arch. Microbiol, 135, 1))]Make OD₆₀₀About 0.1 and incubated at 30 ℃. When the culture reached an OD of about 0.25₆₀₀L-rhamnose (1.0%) was added at that time. Samples (1ml) were taken at different time intervals, centrifuged and the pellet was stored at-20 ℃. Resuspending the pellet in Tris/HCl buffer (b)50mM, pH8.0), the cell suspension was analyzed by SDS-PAGE (FIG. 16).

Example 14

Single chain antibody expression using E.coli secretion system in high cell density fermentation

Coli W3110 was transformed with plasmid pBW 22-pelB-S1. Plasmids were isolated from different clones, verified by restriction analysis, and one clone was used for further experiments. Pre-cultures in shake flasks were inoculated from a single clone in Lonza's batch phase medium. This preculture was inoculated into a 20L fermenter. Cells were grown according to the high cell density fermentation protocol of Lonza. Culture samples (10ml) were obtained at different time points before and after rhamnose induction. Cells were separated from the fermentation medium by centrifugation at 10000 g. SDS gel analysis of samples obtained from cell-free fermentation medium showed a protein band at 28.4kD with increased density. This protein is the single chain antibody S1 released from the growth culture into the fermentation medium. The content of S1 protein was determined by Agilent2100Bioanalyser (Agilent, Palo Alto, USA) and showed an accumulation of up to 2g/L/100OD in the fermentation broth after rhamnose induction₆₀₀S1 protein of (1). After lysozyme treatment of the cell pellet, the insoluble protein pellet contained only traces of S1 protein, while the soluble protein fraction (supernatant) showed a strong S1 protein band, corresponding to about 1g/L/100OD₆₀₀(see FIG. 17).

Claims (19)

1. A vector expressible in a prokaryotic host for expressing an antibody comprising the rhaBAD promoter of the L-rhamnose operon operably linked to a transcription unit and said rhaBAD promoter is represented by SEQ ID NO: 1, the transcription unit contains

a) An antibody-encoding nucleic acid sequence that is heterologous to the host,

b) a prokaryotic signal sequence selected from the group consisting of signal peptides of periplasmatic binding proteins for sugars, amino acids, vitamins and ions, operatively linked to said nucleic acid sequence, the expression of which is controlled by said promoter region, and

c) a translation initiation region located upstream of the translation initiation site of said transcriptional unit, which translation initiation region consists of the sequence aggagatatatacat (SEQ ID NO: 2) wherein said translation initiation region is operably linked to said nucleic acid sequence.

2. The vector of claim 1, wherein the signal peptide is selected from the group consisting of signal peptides from: LamB, MalE, Bla, OppA, TreA, MppA, BglX, ArgT, MalS, HisJ, XylF, FecB, OmpA, and PhoA.

3. The vector of claim 1, wherein said transcriptional unit further comprises a transcription termination region that is a rrnB transcription termination sequence.

4. The vector of claim 1, wherein said nucleic acid sequence encodes a Fab fragment.

5. The vector of claim 4, wherein the heavy and light chains of the Fab fragment are encoded by a dicistronic transcriptional unit.

6. The vector of claim 1, wherein said rhaBAD promoter and said operably linked transcriptional unit are encoded by SEQ ID NO: and 3. forming.

7. The vector of claim 1, wherein said rhaBAD promoter and said operably linked transcriptional unit are encoded by SEQ ID NO: 4.

8. The vector of claim 1, wherein the vector is an autonomously or self-replicating plasmid, cosmid, phage or virus.

9. The vector of claim 8, wherein the virus is a retrovirus.

10. Use of the vector of claim 1 in the regulated heterologous expression of a nucleic acid sequence, wherein said nucleic acid sequence is a nucleic acid sequence encoding an antibody in a prokaryotic host.

11. Use of the vector of claim 10, wherein the antibody is a Fab fragment, the heavy and light chains of which are expressed in equal amounts.

12. An isolated and purified nucleic acid sequence useful for expressing an antibody in a prokaryotic host, comprising the rhaBAD promoter of the L-rhamnose operon operably linked to a transcriptional unit, wherein said rhaBAD promoter is represented by SEQ ID NO: 1, the transcription unit contains

a) An antibody-encoding nucleic acid sequence that is heterologous to the host,

b) a prokaryotic signal sequence selected from the group consisting of signal peptides of periplasmatic binding proteins for sugars, amino acids, vitamins and ions, operatively linked to said nucleic acid sequence, the expression of which is controlled by said promoter region, and

c) the transcription unit further comprises, upstream of its translation initiation site, a translation initiation region consisting of the sequence aggagatataacat (SEQ ID NO: 2) wherein said translation initiation region is operably linked to said nucleic acid sequence.

13. The isolated and purified nucleic acid sequence of claim 12, wherein said rhaBAD promoter and said operably linked transcription unit are encoded by SEQ ID NO: and 3. forming.

14. The isolated and purified nucleic acid sequence of claim 12, wherein said rhaBAD promoter and said operably linked transcription unit are encoded by SEQ ID NO: 4.

15. A prokaryotic host transformed with the vector of claim 1.

16. A prokaryotic host transformed with the isolated and purified nucleic acid sequence of claim 12.

17. A method of producing an antibody in a host, the method comprising the steps of:

a) constructing the vector of claim 1;

b) transforming a prokaryotic host with the vector;

c) under appropriate conditions, allowing the antibody to be expressed in a cell culture system;

d) recovering the antibody from the cell culture system.

18. The method of claim 17, wherein the polypeptide produced is a Fab fragment whose heavy and light chains are expressed in the same amount in the cell culture system.

19. The method of claim 17 or 18, wherein the expression of the antibody is performed in a medium comprising glycerol.