HK1115155B - Melibiose operon expression system - Google Patents

Description

Melibiose operon expression system

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 promoter region of the melibiose operon operably linked to a transcriptional unit comprising a nucleic acid sequence which is heterologous to said host, whereas the expression of this nucleic acid sequence is controlled by said promoter region of the melibiose operon. 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 ] (Yani sch-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. 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. However, many of the known prokaryotic promoters for heterologous gene expression suffer from various disadvantages, such as toxicity of the heterologous product to the host cell, low expression rate of the product, or formation of non-functional aggregates (inclusion bodies).

There are also many inducible promoter systems which have been used for homologous expression of proteins, for example the melibiose operon induced by melibiose as described by Belyaeva et al (2000, mol. mi crobiol., 36(1), 211-222). Such inducible promoter systems for homologous expression mostly have the disadvantage that the rate of induction is low and thus leads to low expression of the desired homologous product. Furthermore, these promoter systems cannot be very tightly regulated, thus generating background activity in an uninduced state, which is not allowed for strict expression control. For example, Belyaeva et al have used different fragments of the melibiose operon fused to the LacZ gene of e.coli (e.coli) to express β -galactosidase in e.coli. However, the fragment producing the highest β -galactosidase activity (KK43, JK19) also produced the highest background activity.

Thus, there remains a need to provide improved prokaryotic expression systems that can be used for heterologous expression of nucleic acid sequences without the above-mentioned disadvantages.

Summary of The Invention

These and other objects, which will be apparent from the foregoing description, are achieved by providing a novel vector comprising a prokaryotic promoter region for high-level expression of a desired heterologous product. Surprisingly, it has been found that the promoter region of the melibiose operon allows tight regulation of the expression of a large number of heterologous products. In a first aspect, the object of the present invention is to provide a novel vector expressible in a host comprising the promoter region of the melibiose operon operably linked to a transcriptional unit comprising a nucleic acid sequence which is heterologous to said host, whereas the expression of said nucleic acid sequence is controlled by said promoter region of melibiose. Also provided are: the use of the novel vectors for the regulated 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 promoter region of the melibiose operon operably linked to a transcriptional unit, wherein said transcriptional unit comprises a nucleic acid sequence which is heterologous to said host, and the expression of said nucleic acid sequence is controlled by said promoter region of the melibiose operon; a prokaryotic host transformed with said vector or said isolated and purified nucleic acid sequence; and a method for producing a polypeptide in a host using the vector.

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 pBLL7 containing the melibiose inducible melAB2 promoter (PmelAB2) and the transcription termination region (rrnB).

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

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

FIG. 4 shows a Western blot of lysozyme extracts of strain W3110(pBLL15) using anti-human light chains for Fab detection coupled to alkaline peroxidase (lane 1: reference Fab-H (2. mu.g); lane 2: W3110(pBLL15), induced, 9 hours; lane 3: W3110(pBLL15), induced, 12 hours; lane 4: W3110(pBLL15), induced, 23 hours).

FIG. 5 shows plasmid pAKL15E containing the melibiose inducible promoter (PmelAB2) and the Fab-H gene with altered signal sequence.

FIG. 6 shows SDS-PAGE of lysozyme extracts of different strains W3110 with high Fab-H antibody concentrations. The strain producing the light and heavy chains without signal sequences was used as a 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. 7 shows plasmid pJKL8 containing the melibiose inducible promoter (PmelAB2) and the sequence of the amidase encoding KIE153 (Burkholderia sp. DSM9925) and the transcription termination region (rrnB).

FIG. 8 shows SDS-PAGE of lysozyme extracts of 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. 9 shows plasmid pBLL7-pelB-S1 containing the melibiose inducible melAB2 promoter and sequences encoding single chain antibodies (scFv, S1). The sequence encoding S1 is preceded by a sequence encoding a PelB signal peptide.

FIG. 10 shows SDS-PAGE of crude extracts of the uninduced (-) and (+) W3110(pBLL7-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.

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 polynucleic acid construct, produced recombinantly or synthetically, with a series of specified polynucleic acid elements that permit transcription of a particular nucleic acid sequence in a host cell. Generally, such vectors include a transcription unit comprising the particular nucleic acid to be transcribed operably linked to a promoter. The vector expressible in the host may be, for example, an autonomously or self-replicating plasmid, a cosmid, a phage, a virus or a retrovirus.

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.

The "melibiose operon" refers to the melibiose operon of E.coli as described by Hanatani et al (1984, J biol. chem., 259(3), 1807-12). The melibiose operon is a positively regulated catabolic operon which contains two divergent promoters. One promoter (melR promoter) is responsible for the expression of the melR gene, which is essential for melibiose-dependent stimulation of the second promoter (melAB promoter). Melibiose-induced transcription from this second promoter initiates the co-transcription of the melA gene encoding alpha-galactosidase and the melB gene encoding melibiose permease. The melibiose operon contains two catabolite Regulator Protein (catabolite Regulator Protein) binding sites: CRP2 at position-81, 5 and CRP1 at position-195, 5 upstream of the transcription start site of the melAB promoter; and 5 MelR binding sites, respectively-42, 5 (position 2 '), -62, 5 (position 2), -100, 5 (position 1), -120, 5 (position 1'), -238, 5 (position R) upstream of the transcription initiation site of the MelAB promoter. The "promoter region of melibiose operon" refers to a promoter region regulating the expression of the melA gene and the melB gene, and includes the melAB promoter, the melR gene and CRP binding site, and the MelR binding site. The "melAB promoter" herein essentially consists of a transcription promoter site, a putative-35 region, a Pribnow box, a CRP binding site and a MelR binding site. The "melAB promoter deficient in the CRP1 binding site" herein essentially consists of the transcription initiation site, the putative-35 region, the Pribnow box, the CRP2 binding site and the MelR binding site, without site R. The "melAB promoter deficient in the CRP1 binding site" may also contain a CRP1 binding site which is inhibited or blocked. This can be achieved using known techniques, such as transposon-supported mutagenesis or knockout mutations. Preferably, the "melAB promoter deficient in the CRP1 binding site" does not contain any CRP1 binding site.

Melibiose (6-O- [ alpha ] -D-galactosylpyranyl-D-glucose) is a disaccharide obtainable by fermentation of raffinose with yeast.

"CRP" means a "catabolite regulatory 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) that mediates the activation of catabolic operons such as the melibiose operon.

An "enhancer" is a nucleic acid sequence that functions to enhance transcription of a transcriptional unit independent of identity to 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 translation initiation region, such as a ribosome binding site, is operably linked to a nucleic acid sequence encoding a polypeptide if it is positioned so as to facilitate translation of the polypeptide. 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 such as 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 sequences will be free or substantially free of materials with which they are naturally associated, such as other nucleic acids with which they are associated as found in their natural environment or in their environment of manufacture (e.g., cell culture) when such articles are made using recombinant techniques, whether 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" of the kind 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 Autobel 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 that encodes a short amino acid sequence (i.e., a signal peptide) that is present at the NH 2-terminus of certain proteins that are normally transported by cells to non-cytoplasmic locations (e.g., secretion) or as membrane components. 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 κ 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 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 region 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 invention provides a vector expressible in a host comprising the promoter region of the melibiose operon operably linked to a transcriptional unit comprising a nucleic acid sequence which is heterologous to said host, whereby the expression of this nucleic acid sequence is controlled by said promoter region of the melibiose operon.

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 pBLL15 or pAKL 15E. 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, El sevi er, Amsterdam-New York-Oxford, 1985).

Preferred vectors of the invention are autonomously or self-replicating plasmids, more preferably vectors with a specific host range, such as vectors specific for e.g.E.coli. Most preferred are pBR322, pUC18, pACYC177, pACYC184, pRSF1010 and pBW22 or derivatives thereof (such as plasmid pBLL15 or pAKL15E), especially preferred are pBW22 or derivatives thereof (such as plasmid pBLL15 or pAKL15E), more especially preferred are pBLL15 or pAKL15E, and most preferred are pAKL 15E.

In a preferred embodiment, the promoter region of the melibiose operon used in the present invention is the melAB promoter. According to a more preferred embodiment of the invention said melAB promoter is deficient in the CRP1 binding site. In a particularly preferred embodiment, the melAB promoter lacking the CRP1 binding site consists of SEQ ID NO: l, its complementary sequence or variant. Typically, the MelR binding site of the promoter region is not modified. The promoter region of the melibiose operon used in the present invention, the melAB promoter lacking the CRP1 binding site, and the promoter region consisting of SEQ ID NO: 1. the melAB promoter deficient in the CRP1 binding site, constituted by its complementary sequence or variant, is usually obtained from the melibiose operon of E.coli, or from a functionally equivalent promoter region of other prokaryotes, in particular of organisms of the Enterobacteriaceae family (Enterobacteriaceae). Preferably, the promoter region of the melibiose operon, the melAB promoter lacking the CRP1 binding site, and the melAB promoter consisting of SEQ ID NO. 1, its complementary sequence or variant lacking the CRP1 binding site are derived from the melibiose operon of E.coli. Functionally equivalent promoter regions of other prokaryotes include promoter regions inducible by melibiose, i.e.promoter regions whose expression activity is higher in the presence of melibiose than in the absence of melibiose.

The transcription unit of the invention typically further comprises a translation initiation region upstream of the translation initiation site of said transcription unit, said translation initiation region consisting of 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 immediately adjacent to the translation start site (typically ATG, GTG or TTG) of the transcription unit.

Typically, the transcriptional unit further comprises a signal sequence operably linked to the heterologous nucleic acid sequence of the invention. Where a dicistronic or polycistronic transcription unit is used, different or identical signal sequences may be operably linked to each cistron. In this case, different signal sequences are preferably used. The signal sequence used may be a prokaryotic or eukaryotic signal sequence. Usually, prokaryotic signal sequences are used. Eukaryotic signal sequences which can be used and are particularly useful in E.coli are, for example, the signal sequence of human ceruloplasmin, the signal sequence of the human neutrophil defensin 1, 2, 3 precursor or the signal peptide of the arylsulfatase of Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) described in WO 03068956. Prokaryotic signal sequences commonly used are signal peptides of periplasmic (periplamatic) binding proteins for sugars, amino acids, vitamins and ions, such as PelB (erwinia chrysanthemi (erwiniacechrysogeneci), pectate lyase precursor), PelB (erwinia carotovora), pectate lyase precursor), PelB (xanthomonas campestris), pectate lyase precursor), LamB (escherichia coli, maltoporin protein 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), mbppa (escherichia coli, periplasmic peptide binding protein precursor), bglxx (escherichia coli, periplasmic beta-glucosidase precursor), lysine, ArgT (escherichia coli, arginin-ornithine binding periplasmic protein precursor), e, MalS (e.coli, a-amylase precursor), Hi sJ (e.coli, histidine-binding periplasmic protein precursor), XylF (e.coli, D-xylose-binding periplasmic protein precursor), FecB (e.coli, dicitrate-binding 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), Hi sJ (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). Particularly preferred are the E.coli signal sequences LamB (maltoporin precursor) and MalE (maltose-binding protein precursor).

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).

Typically, the transcriptional unit further comprises a transcriptional termination region selected from the group consisting of rrnB, RNAI, T7Te, rrnB T1, trp a L126, trp a, tR2, T3Te, P14, tonB T, and trp a L153. Preferably, the rrnB transcription termination sequence is 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. 5,401,629 and 5,436,128). Other proteins of interest are adhesion proteins, such as integrins), selectins (selecting), members of the immunoglobulin superfamily (see Springer, Nature346, 425-; 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 of the Fab fragment are encoded by a dicistronic transcriptional unit, whereas each chain is operably linked to a signal sequence 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 of the invention consist of the sequences SEQ id no: 3. its complementary sequence and its variant.

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

The invention also includes the use of the vectors of the invention to regulate the heterologous expression of nucleic acid sequences in prokaryotic hosts. Expression can be regulated by the amount of melibiose available to the prokaryotic host. In general, the amount of melibiose in the culture medium of the cultured prokaryotic host is between 0.01 and 100g/l, preferably between 0.1 and 10g/l, more preferably between 1 and 5 g/l.

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

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 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 (e.g.lysozyme extract of cultured host cells).

In another aspect, the invention provides an isolated and purified nucleic acid sequence for expression in a host, said sequence comprising the promoter region of the melibiose operon operably linked to a transcriptional unit comprising a nucleic acid sequence which is heterologous to said host, whereby expression of said nucleic acid sequence is controlled by said promoter region of the melibiose operon. The melAB promoter is a preferred promoter region. More preferred is the melAB promoter lacking the CRP1 binding site. Most 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 known in the art, the isolated and purified DNA sequence may also contain one or more regulatory sequences, such as enhancers, which are normally 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 is to provide a prokaryotic host transformed with a vector of the invention. In a particular embodiment of the invention, the prokaryotic host is transformed with plasmid pBLL15 or pAKL15E, preferably plasmid pAKL15E containing two different coding regions in its dicistronic expression cassette, for expression of secreted heterodimeric proteins such as Fab in the host cell. Preferably, such dimeric protein is a Fab. In another embodiment of the invention, a prokaryotic host of the invention 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 melibiose. Host cells that are normally capable of ingesting and metabolizing melibiose (e.g., E.coli) may be modified to lack one or more functions associated with ingesting and/or metabolizing melibiose. Defects in one or more functions associated with the uptake and/or metabolism of melibiose can be achieved, for example, by inhibiting or blocking a gene encoding a protein associated with the uptake and/or metabolism of melibiose (e.g., the melA gene encoding alpha-galactosidase). 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 culture may be used in culture tubes, shake flasks or bacterial fermentors, such as batch or fed-batch culture. 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 melibiose in order to achieve a tight regulation of the melibiose promoter region. Usually the appropriate OD is achieved in the culture medium₆₀₀(depending on the culture system) melibiose was added afterwards. In general, the amount of melibiose in the medium in which the prokaryotic host is cultured is between 0.01 and 100g/l, preferably between 0.1 and 10g/l, more preferably between 1 and 5 g/l. For batch culture, the usual OD₆₀₀Typically 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, such as 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.

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 expressible in a prokaryotic host and comprises the promoter region of the melibiose operon operably linked to a transcriptional unit comprising a nucleic acid sequence which is heterologous to said host. Since in embodiments of the vectors 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 regulating the 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 the vector is useful for intracellular expression, the promoter region of the melibiose operon is the melAB promoter, which is preferably deficient in the CRP1 binding site. It is particularly preferred that the melAB promoter lacking the CRP1 binding site consists of the amino acid sequence of SEQ ID NO:1, its complement and variants thereof. 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

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

construction of expression plasmids with positively regulated promoters:

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 at the corresponding regulator binding site. Chromosomal DNA of E.coli was isolated by the method described by Pitcher et al (1989, Letters in Applied Microbiology8, 151-156). The promoter fragment was amplified from the chromosomal DNA of strain W3110 using PCR with 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 tatttg3’

Pprp-3 5’aaa ctt aag ttg tta tca act tgt tat3’

AAAATCGATAACTGAAACGCATATTTGCGGATTAGTTCATGACTTTATCTCTAACAAA

TTGAAATTAAACATTTAATTTTATTAAGGCAATTGTGGCACACCCCTTGCTTTGTCTTT

ATCAACGCAAATAACAAGTTGATAACAACTTAAGTTT

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

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

AAAATCGATGCATCACGCCCCGCACAAGGAAGCGGTAGTCACTGCCCGATACGGAC

TTTACATAACTCAACTCATTCCCCTCGCTATCCTTTTATTCAAACTTTCAAATTAAAATA

TTTATCTTTCATTTTGCGATCAAAATAACACTTTTAAATCTTTCAATCTGATTAGATTAG

GTTGCCGTTTGGTAATAAAACAATAAATCCTGACTTAAGTTT

PmelAB2 PmelAB-5-1 5’aaaatc gatgactgcgag tgg gag cac3′

PmelAB-3 5′aaactt aagggcttg ctt gaa taa ctt3’

MelR CRP

AAAATCGATATT

(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 polypeptide comprising a sequence consisting of SEQ ID NO: a plasmid (pBLL7) comprising the melAB2 promoter of FIG. 1 is shown in FIG. 1. The resulting plasmid containing the prp promoter (pBLL5) and the plasmid containing the gutA promoter (pBLL6) were identical except for the promoter region to which they were ligated. The sequence of the inserted promoter was determined by sequencing (Microsynth GmbH, Balgach, Switzerland).

Example 2

Construction of Fab fragment expression plasmid

As an alternative to the IPTG-inducible lac promoter (plasmid pMx9-HuCAL-Fab-H, Knappik et al, 1985, Gene33, 103-119), the ability of different positive regulated expression systems to produce Fab-H antibody fragments was analyzed. 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 to generate plasmid pBW22-Fab-H containing the L-rhamnose inducible rhaBAD promoter (Volff et al, 1996, mol. Microbiol.22, 1037-1047). The same PCR fragment was inserted into a different expression plasmid with an inducible promoter. The resulting (putative) expression plasmid pBLL15 containing the Fab-H and melAB2 promoter (SEQ ID NO: 3) is shown in FIG. 2. Equivalent plasmids containing the prp promoter (pBLL13) and the gutA promoter (pBLL14) were obtained. 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 (DSM5911, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) was transformed with different expression plasmids. 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 reisolated 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 auto-folded Fab-H antibody fragments. This yield test was performed as described in example 4. The following elicitors 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 L-rhamnose and melibiose induced strains 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 TG 1F' - (pMx9-HuCAL-Fab-H) were used as controls. The concentration of Fab-H was measured in mg/100OD/L (nd means not measured).

All strains grew well in the presence or absence of the corresponding elicitor, without growth inhibition, DO₆₀₀To between 4 and 6. Expression plasmids pBW22-Fab-H 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 L-rhamnose (pBW22-Fab-H) induced system.

The L-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 ℃. The frozen cells were lysed according to the lysozyme treatment described above and analyzed in dot blot and ELISA assaysAnd (4) supernatant fluid. 504.28mg/L/100OD was obtained₆₀₀A functional Fab-H antibody fragment of (1).

Example 5

Presence 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. Western blotting of lysozyme extract of strain W3110(pBLL15) using the anti-human Fab-H + AP conjugate is shown in FIG. 4. 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 in which antibodies are formed: functional 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 relatively homogeneous (homogeneous) group that has 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, into the cytoplasm. The Si gnalP website server (http:// www.cbs.dtu.dk/services/SignalP-2.0/# transmission) was used to check the sequence peptide and dissociation site probabilities for all possible signal peptide Fab combinations, 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 a single peptide sequence to VL3-CL and VH-CH sequences ((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 partitioning byaverap extension. engineered hybrid genes without restriction enzymes: Gene77, 61-68 obtained by overlap extension.) splicing SP-VL3-CL genes with restriction enzymes NdeI and PstI and into NdeI/PstI spliced pBw22 and pBLL7 were performed as described elsewhere.

The resulting plasmid was cut with PstI and HindIII and ligated to the PstI/HindIII cut SP-VH-CH gene. Since integration of bla-VL3-CL and fecB-VH-CH genes was not possible, only Fab-H expression plasmids containing the lamB-VL3-CL and malE-VH-CH genes were tested. A lamB-VL3-CL/malE-VH-CH expression plasmid containing the L-rhamnose inducible promoter was obtained (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. FIG. 5 illustrates the expression plasmid pAKL15E (Seq ID No.4) containing the melibiose inducible promoter and lamB-VL 3-CL/malE-VH-CH.

Example 7

Effect of translation initiation region on Fab expression

The Fab-H genes of plasmid pAKL14 and plasmid pAKL15E contained the same DNA sequence 5' 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.

New signal peptide constructs (in combination with modified translation initiation signals) again antibodyThe fragment titer was 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-CL3-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

GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKKVEPKS

the light chain of Fab-H has two errors (D50N and K63N) and the heavy chain has one amino acid exchange (F156L). To preserve the original Fab-H sequence, two fragments of plasmid pAKL14 (138bp SexAI/BamHI and 310bp BssHII/HindIII fragments) 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 had no 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 yield of Fab-H antibody fragments can be increased by using different optimization strategies. Table 5 below summarizes these improvements:

bacterial strains	Improvements in or relating to	Concentration of functional Fab-H antibody (mg/L/100OD)	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: (M))	878，86	10.4

Strains producing high Fab-H antibody titers were analyzed by SDS-PAGE (FIG. 6). The highest functional Fab-H concentration was measured in strains producing balanced amounts of light and heavy chains (lanes 4 and 5). L-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 carrying plasmid pAKL15E was tested for its ability to produce auto-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 lysozyme treatment as described above 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 5). The light and heavy chains of Fab-H were produced in equal amounts (FIG. 8).

Example 9

Melibiose Induction of amidase from strain KIE153 (Burkholderia sp. DSM9925) in Shake flasks

Coli W3110 (FIG. 7) carrying pJKL8 was tested for its ability to convert racemic piperazine-2-carboxamide to (R) -piperazine-2-carboxylic acid. The overnight culture [ in NYB medium (10g/l tryptone, 5g/l yeast extract, 5g/l sodium chloride) was diluted (1: 50) in 20ml of 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)) The medium was supplemented with 100. mu.g/ml ampicillin, 30 ℃ C]And incubated at 30 ℃. When the culture reached an OD of about 0.8₆₀₀Melibiose (0.2%) was added at this time. Cells were harvested 19 hours after induction and stored at-20 ℃. Amidase activity was tested as described by Eichhorn et al (1997, tetrahedron asymmetry, 8(15), 2533-36). Resting cells at about 1g/h/OD₆₀₀The conversion rate of (a) enantioselectively converts (R) -piperazine-2-carboxamide into (R) -piperazine-2-carboxylic acid.

Example 10

Melibiose 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 pBLL7 to generate plasmid pBLL7-pelB1-S1 containing the melibiose inducible melAB2 promoter (FIG. 9). The S1 insert sequence of plasmid pBLL7-S1 was determined by sequencing. Strain W3110 (DSM5911, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) was transformed with plasmid pBLL 7-pelBl-S1. Plasmids were isolated from different clones and verified by restriction analysis. Coli W3110(pBLL7-pelB-S1) was tested for its ability to produce soluble S1. The overnight cultures [ in NYB medium (10g/l tryptone, 5g/l yeast extract, 5g/l sodium chloride) supplemented with 100. mu.g/ml ampicillin, 37 ℃ C. (Biotechnol. 39, 59-65) were diluted (1: 50) in 20ml fresh glycerol medium (as described by Kortz et al, 1995, J.Biotechnol.39, 59-65, except where the vitamin solution was different (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 lysozyme treatment as described above, and supernatants were analyzed by SDS-PAGE and BioanalyzerInsoluble protein precipitate. Most of the S1 protein (1.7g/Lx mg/100 OD)₆₀₀) Produced in the form of a soluble protein fraction.

Sequence listing

<110> Longsa-stockings company (LONZA AG)

Mofosisis shares company (MORPHOSYS AG)

<120> melibiose operon expression System

<130>B14868/EP

<160>4

<170>PatentIn version 3.3

<210>1

<211>143

<212>DNA

<213> Escherichia coli (Escherichia coli)

<220>

<221>misc_feature

<223> melAB2 promoter

<400>1

actctgcttt tcaggtaatt tattcccata aactcagatt tactgctgct tcacgcagga 60

tctgagttta tgggaatgct caacctggaa gccggaggtt ttctgcagat tcgcctgcca 120

tgatgaagtt attcaagcaa gcc 143

<210>2

<211>13

<212>DNA

<213> Escherichia coli (Escherichia coli)

<220>

<221>misc_feature

<223> translation initiation region

<400>2

aggagatata cat 13

<210>3

<211>1630

<212>DNA

<213> Escherichia coli (Escherichia coli)

<220>

<221>misc_feature

<223> sequence containing melAB2 promoter and ompA-VL3-CL and phoA-VH-CH

<400>3

atcgatactc tgcttttcag gtaatttatt cccataaact cagatttact gctgcttcac 60

gcaggatctg agtttatggg aatgctcaac ctggaagccg gaggttttct gcagattcgc 120

ctgccatgat gaagttattc aagcaagccc ttaagaagga gatatacata tgaaaaagac 180

agctatcgcg attgcagtgg cactggctgg tttcgctacc gtagcgcagg ccgatatcga 240

actgacccag ccgccttcag tgagcgttgc accaggtcag accgcgcgta tctcgtgtag 300

cggcgatgcg ctgggcgata aatacgcgag ctggtaccag cagaaacccg ggcaggcgcc 360

agttctggtg atttatgatg attctgaccg tccctcaggc atcccggaac gctttagcgg 420

atccaacagc ggcaacaccg cgaccctgac cattagcggc actcaggcgg aagacgaagc 480

ggattattat tgccagagct atgactctcc tcaggttgtg tttggcggcg gcacgaagtt 540

aaccgttctt ggccagccga aagccgcacc gagtgtgacg ctgtttccgc cgagcagcga 600

agaattgcag gcgaacaaag cgaccctggt gtgcctgatt agcgactttt atccgggagc 660

cgtgacagtg gcctggaagg cagatagcag ccccgtcaag gcgggagtgg agaccaccac 720

accctccaaa caaagcaaca acaagtacgc ggccagcagc tatctgagcc tgacgcctga 780

gcagtggaag tcccacagaa gctacagctg ccaggtcacg catgagggga gcaccgtgga 840

aaaaaccgtt gcgccgactg aggcctgata agcatgcgta ggagaaaata aaatgaaaca 900

aagcactatt gcactggcac tcttaccgtt gctcttcacc cctgttacca aagcccaggt 960

gcaattgaaa gaaagcggcc cggccctggt gaaaccgacc caaaccctga ccctgacctg 1020

taccttttcc ggatttagcc tgtccacgtc tggcgttggc gtgggctgga ttcgccagcc 1080

gcctgggaaa gccctcgagt ggctggctct gattgattgg gatgatgata agtattatag 1140

caccagcctg aaaacgcgtc tgaccattag caaagatact tcgaaaaatc aggtggtgct 1200

gactatgacc aacatggacc cggtggatac ggccacctat tattgcgcgc gttatcctgt 1260

tactcagcgt tcttatatgg atgtttgggg ccaaggcacc ctggtgacgg ttagctcagc 1320

gtcgaccaaa ggtccaagcg tgtttccgct ggctccgagc agcaaaagca ccagcggcgg 1380

cacggctgcc ctgggctgcc tggttaaaga ttatttcccg gaaccagtca ccgtgagctg 1440

gaacagcggg gcgctgacca gcggcgtgca tacctttccg gcggtgctgc aaagcagcgg 1500

cctgtatagc ctgagcagcg ttgtgaccgt gccgagcagc agcttaggca ctcagaccta 1560

tatttgcaac gtgaaccata aaecgagcaa caccaaagtg gataaaaaag tggaaccgaa 1620

aagctgataa 1630

<210>4

<211>1659

<212>DNA

<213> Escherichia coli (Escherichia coli)

<220>

<221>misc_feature

<223> sequence containing melAB2 promoter and lamB-VL3-CL and malE-VH-CH

<400>4

atcgatactc tgcttttcag gtaatttatt cccataaact cagatttact gctgcttcac 60

gcaggatctg agtttatggg aatgctcaac ctggaagccg gaggttttct gcagattcgc 120

ctgccatgat gaagttattc aagcaagccc ttaagaagga gatatacata tgatgattac 180

tctgcgcaaa cttcctctgg cggttgccgt cgcagcgggc gtaatgtctg ctcaggcaat 240

ggctgatatc gaactgaccc agccgccttc agtgagcgtt gcaccaggtc agaccgcgcg 300

tatctcgtgt agcggcgatg cgctgggcga taaatacgcg agctggtacc agcagaaacc 360

cgggcaggcg ccagttctgg tgatttatga tgattctgac cgtccctcag gcatcccgga 420

acgctttagc ggatccaaca gcggcaacac cgcgaccctg accattagcg gcactcaggc 480

ggaagacgaa gcggattatt attgccagag ctatgactct cctcaggttg tgtttggcgg 540

cggcacgaag ttaaccgttc ttggccagcc gaaagccgca ccgagtgtga cgctgtttcc 600

gccgagcagc gaagaattgc aggcgaacaa agcgaccctg gtgtgcctga ttagcgactt 660

ttatccggga gccgtgacag tggcctggaa ggcagatagc agccccgtca aggcgggagt 720

ggagaccacc acaccctcca aacaaagcaa caacaagtac gcggccagca gctatctgag 780

cctgacgcct gagcagtgga agtcccacag aagctacagc tgccaggtca cgcatgaggg 840

gagcaccgtg gaaaaaaccg ttgcgccgac tgaggcctga taactgcagg agatatacat 900

atgaaaataa aaacaggtgc acgcatcctc gcattatccg cattaacgac gatgatgttt 960

tccgcctcgg ctctcgccca ggtgcaattg aaagaaagcg gcccggccct ggtgaaaccg 1020

acccaaaccc tgaccctgac ctgtaccttt tccggattta gcctgtccac gtctggcgtt 1080

ggcgtgggct ggattcgcca gccgcctggg aaagccctcg agtggctggc tctgattgat 1140

tgggatgatg ataagtatta tagcaccagc ctgaaaacgc gtctgaccat tagcaaagat 1200

acttcgaaaa atcaggtggt gctgactatg accaacatgg acccggtgga tacggccacc 1260

tattattgcg cgcgttatcc tgttactcag cgttcttata tggatgtttg gggccaaggc 1320

accctggtga cggttagctc agcgtcgacc aaaggtccaa gcgtgtttcc gctggctccg 1380

agcagcaaaa gcaccagcgg cggcacggct gccctgggct gcctggttaa agattatttc 1440

ccggaaccag tcaccgtgag ctggaacagc ggggcgctga ccagcggcgt gcataccttt 1500

ccggcggtgc tgcaaagcag cggcctgtat agcctgagca gcgttgtgac cgtgccgagc 1560

agcagcttag gcactcagac ctatatttgc aacgtgaacc ataaaccgag caacaccaaa 1620

gtggataaaa aagtggaacc gaaaagctga taaaagctt 1659

Claims (25)

1. A vector expressible in a prokaryotic host comprising the melAB promoter of the melibiose operon lacking the CRP1 binding site operably linked to a transcriptional unit comprising a nucleic acid sequence which is heterologous to said host, the expression of said nucleic acid sequence being controlled by said promoter, wherein said melAB promoter lacking the CRP1 binding site consists of the amino acid sequence of SEQ ID NO:1 or the complement thereof.

2. The vector of claim 1, wherein said transcriptional unit further comprises, upstream of the initiation site of translation of said transcriptional unit, a translation initiation region consisting of the sequence aggagagatataacat, SEQ ID NO: 2, and the translation initiation region is operably linked to the nucleic acid sequence.

3. The vector of claim 1, wherein said transcription unit further comprises a signal sequence operably linked to said nucleic acid sequence.

4. The vector of claim 3, wherein the signal sequence is a prokaryotic signal sequence.

5. The vector of claim 4, wherein said prokaryotic signal sequence is selected from the group consisting of nucleic acid sequences encoding signal peptides of periplasmatic binding proteins for sugars, amino acids, vitamins and ions.

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

7. The vector of claim 1, wherein said nucleic acid sequence encodes a polypeptide.

8. The vector of claim 1, wherein the nucleic acid sequence encodes an antibody.

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

10. The vector of claim 9, wherein the heavy and light chains of said Fab fragment are encoded by a dicistronic transcriptional unit, each chain being operably linked to a signal sequence and the same translation initiation region upstream of the initiation site of translation of said transcriptional unit.

11. The vector of claim 1, wherein said promoter and said operably linked transcription unit consist of SEQ ID NO: 3 or the complement thereof.

12. The vector of claim 1, wherein said promoter and said operably linked transcription unit consist of SEQ ID NO: 4 or the complement thereof.

13. The vector of claim 1, wherein the vector is an autonomously replicating plasmid, a cosmid, a phage, a virus or a retrovirus.

14. The vector of claim 1, wherein the vector is a self-replicating plasmid.

15. Use of the vector of any one of claims 1-14 for the regulation of heterologous expression of a nucleic acid sequence in a prokaryotic host.

16. Use of the vector of claim 15, wherein the nucleic acid sequence encodes a polypeptide.

17. Use of the vector of claim 16, wherein the polypeptide is a Fab fragment and the heavy and light chains of the Fab fragment are expressed in equal amounts.

18. An isolated and purified nucleic acid expressible in a prokaryotic host comprising the melAB promoter of the melibiose operon lacking the CRP1 binding site operably linked to a transcriptional unit comprising a nucleic acid sequence which is heterologous to said host, the expression of said nucleic acid sequence being controlled by said promoter, wherein said melAB promoter lacking the CRP1 binding site is represented by SEQ ID NO:1 or the complement thereof.

19. The isolated and purified nucleic acid of claim 18, wherein the promoter and the operably linked transcription unit are defined by SEQ ID NO: 3 or the complement thereof.

20. The isolated and purified nucleic acid of claim 18, wherein the promoter and the operably linked transcription unit are defined by SEQ ID NO: 4 or the complement thereof.

21. A prokaryotic host transformed with the vector of any one of claims 1-14.

22. A prokaryotic host transformed with the isolated and purified nucleic acid of any one of claims 18-20.

23. A method of producing a polypeptide in a host, the method comprising the steps of:

a) constructing the vector of any one of claims 1-14;

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.

24. The method of claim 23, wherein the polypeptide produced is a Fab fragment whose heavy and light chains are expressed in the same amount in the cell culture system.

25. The method of claim 23 or 24, wherein expression of the polypeptide is performed in a medium comprising glycerol.