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United States Patent Application 20180057823
Kind Code A1
Kara; Bhupendra Vallabh ;   et al. March 1, 2018

Expression Vector

Abstract

An expression vector for expressing a target polypeptide in a prokaryotic cell is provided. The vector comprises a promoter operably linked to a polynucleotide encoding the target polypeptide operably linked to a eukaryotic secretion leader sequence, the eukaryotic secretion leader sequence encoding a signal peptide sequence selected from the group consisting of: a) MLKRSSWLATLGLLTVASVSTIVYA; b) MKKATFITCLLAVLLVSNPIWNA; c) MKVSAAALAVILIATALCAPASA; d) MKVSTAFLCLLLTVSAFSAQVLA; and e) MKCLLLALGLALACAAQA. Processes for expressing polypeptides and prokaryotic microorganisms comprising such vectors are also provided.


Inventors: Kara; Bhupendra Vallabh; (Billingham, UK) ; Lennon; Christopher David John; (Billingham, UK)
Applicant:
Name City State Country Type

Fujifilm Diosynth Biotechnologies UK Limited

Billingham

GB
Assignee: Fujifilm Diosynth Biotechnologies UK Limited
Billingham
GB

Family ID: 1000002983238
Appl. No.: 15/813436
Filed: November 15, 2017


Related U.S. Patent Documents

Application NumberFiling DatePatent Number
14880671Oct 12, 20159845475
15813436
12995924Dec 2, 20109157085
PCT/GB2009/001372Jun 1, 2009
14880671

Current U.S. Class: 1/1
Current CPC Class: C12N 15/625 20130101; C12N 15/70 20130101
International Class: C12N 15/70 20060101 C12N015/70; C12N 15/62 20060101 C12N015/62

Foreign Application Data

DateCodeApplication Number
Jun 4, 2008GB0810154.5

Claims



1-14. (canceled)

15. A prokaryotic expression vector comprising a prokaryotic promoter operably linked to a heterologous polynucleotide encoding a target polypeptide operably linked to a eukaryotic secretion leader, the eukaryotic secretion leader encoded by the polynucleotide being a signal peptide having an amino acid sequence at least 90% identical to SEQ ID NO: 2, 3 or 5.

16. The prokaryotic expression vector according to claim 15, wherein the signal peptide has an amino acid sequence at least 95% identical with SEQ ID NO: 2, 3 or 5.

17. The prokaryotic expression vector according to claim 15, wherein the polynucleotide encoding a target polypeptide operably linked to a eukaryotic secretion leader sequence has structure such that the polynucleotide encoding the eukaryotic secretion leader sequence is attached at the 5' end of the polynucleotide encoding the target polypeptide.

18. The prokaryotic expression vector according to claim 15, wherein the vector is a plasmid.

19. The prokaryotic expression vector according to claim 15, wherein the target polypeptide is selected from the group consisting of cytokines, growth factors, antibodies, antibody fragments, immunoglobulin like polypeptides, enzyme, vaccines, peptide hormones, chemokines, receptors, receptor fragments, kinases, phosphatases, isomerases, hydrolyases, transcription factors and fusion polypeptides.

20. A method for the production of a target polypeptide which comprises expressing the prokaryotic expression vector according to claim 15 in a prokaryotic host cell.

21. The method according to claim 20, wherein the prokaryotic host cell is selected from the group consisting of E. coli, Salmonella typhimurium, Serratia marsescens, Pseudomonas aeruginosa, Pseudomonas putida and Bacillus subtilis.

22. A prokaryotic microorganism comprising the prokaryotic expression vector according to claim 15.

23. The prokaryotic microorganism according to claim 22, wherein the microorganism is selected from the group consisting of E. coli, Salmonella typhimurium, Serratia marsescens, Pseudomonas putida, Pseudomonas aeruginosa, and Bacillus subtilis.

24. A method for producing a target polypeptide which comprises: a) culturing a prokaryotic host cell comprising the prokaryotic expression vector according to claim 15, thereby to express the target polypeptide, and b) recovering the target polypeptide.

25. The method according to claim 24, wherein the polynucleotide encoding the target polypeptide operably linked to the eukaryotic secretion leader sequence has structure such that the polynucleotide encoding the eukaryotic secretion leader sequence is attached at the 5' end of the polynucleotide encoding the target polypeptide.

26. The method according to claim 24, wherein the target polypeptide is selected from the group consisting of cytokines, growth factors, antibodies, antibody fragments, immunoglobulin like polypeptides, enzyme, vaccines, peptide hormones, chemokines, receptors, receptor fragments, kinases, phosphatases, isomerases, hydrolyases, transcription factors and fusion polypeptides.

27. The method according to claim 24, wherein the signal peptide has the sequence of SEQ ID NO: 2, 3 or 5.

28. The method according to claim 24, wherein the prokaryotic host cell is selected from the group consisting of E. coli, Salmonella typhimurium, Serratia marsescens, Pseudomonas aeruginosa, Pseudomonas putida and Bacillus subtilis.

29. The method according to claim 28, wherein the signal peptide has the sequence of SEQ ID NO: 2, 3 or 5.

30. The method according to claim 24, wherein the prokaryotic promoter is an E. coli polymerase-based promoter.

31. The prokaryotic expression vector according to claim 15, wherein the signal peptide has the amino acid sequence at least 90% identical with SEQ ID NO: 2.

32. The prokaryotic expression vector according to claim 15, wherein the signal peptide has the amino acid sequence at least 90% identical with SEQ ID NO: 3.

33. The prokaryotic expression vector according to claim 15, wherein the signal peptide has the amino acid sequence at least 90% identical with SEQ ID NO: 5.

34. The prokaryotic expression vector according to claim 15, wherein the signal peptide has the amino acid sequence of SEQ ID NO: 2, 3 or 5.
Description



SEQUENCE LISTING SUBMISSION VIA EFS-WEB

[0001] A computer readable text file, entitled "SequenceListing.txt," created on or about Nov. 14, 2017 with a file size of about 14 kb contains the sequence listing for this application and is hereby incorporated by reference in its entirety.

[0002] The present invention concerns a process for the expression of a polypeptide in a prokaryotic cell using eukaryotic secretion leader sequences.

[0003] It is of significant benefit in recombinant polypeptide production if the polypeptide of interest can be exported from the cell in which it is expressed. Expression systems are therefore advantageously designed to enable such export, or secretion. Secretion of the recombinant polypeptide from the host cell commonly involves use of signal peptides, which are found on the majority of eukaryotic and prokaryotic proteins that are destined for export from the cytoplasm. Signal peptides employed in such expression systems are typically native to the expression host, for example, the PhoA, MaIB and OmpA signal peptides of Escherichia coli have been used extensively to secrete polypeptides to the periplasm of that organism. As a matter of course, the use of prokaryotic hosts involves the use of prokaryotic signal peptides. Prokaryotic secretion leader sequences encoding suitable signal peptides are therefore commonly included in prokaryotic expression systems.

[0004] The expression of eukaryotic proteins using prokaryotic expression hosts often leads to highly unpredictable and inconsistent secretion of recombinant polypeptides. The use of many eukaryotic signal peptides in different systems results in expression systems which are inefficient, with low yields being commonplace. In addition, problems may be encountered with the misprocessing of the signal peptide, which may be improperly removed or incompletely cleaved. Thus there is a need for eukaryotic secretion signal peptides that result in efficient expression and secretion of recombinant polypeptides in prokaryotic hosts.

BRIEF DESCRIPTION OF DRAWINGS

[0005] FIG. 1 shows SDS-PAGE analysis results of shake-flask evaluation of Strain 1.

[0006] FIG. 2 shows SDS-PAGE analysis results of shake-flask evaluation of Strain 2.

[0007] FIG. 3 shows SDS-PAGE analysis results of shake-flask evaluation of Strain 3.

[0008] FIG. 4 shows SDS-PAGE analysis results of shake-flask evaluation of Strains 5 and 6.

[0009] FIG. 5 shows SDS-PAGE analysis results of shake-flask evaluation of Strains 7 and 8.

[0010] FIG. 6 shows SDS-PAGE analysis results of fermentation analysis of Strain 1.

[0011] FIG. 7 shows SDS-PAGE analysis results of fermentation analysis of Strain 3.

[0012] FIG. 8 depicts secretion of thioredoxin by Strains 9, 10 and 11.

[0013] FIG. 9 depicts secretion of human growth hormone by Strain 12.

[0014] FIG. 10 depicts secretion of thioredoxin by Pseudomonas putida.

[0015] According to one aspect of the present invention, there is provided an expression vector for expressing a target polypeptide in a prokaryotic cell, comprising a promoter operably linked to a polynucleotide encoding a target polypeptide operably linked to a eukaryotic secretion leader sequence, the eukaryotic secretion leader sequence encoding a signal peptide sequence selected from the group consisting of:

TABLE-US-00001 a) (SEQ ID NO: 1) MLKRSSWLATLGLLTVASVSTIVYA; b) (SEQ ID NO: 2) MKKATFITCLLAVLLVSNPIVVNA; c) (SEQ ID NO: 3) MKVSAAALAVILIATALCAPASA; d) (SEQ ID NO: 4) MKVSTAFLCLLLTVSAFSAQVLA; and e) (SEQ ID NO: 5) MKCLLLALGLALACAAQA

[0016] or a functional equivalent thereof.

[0017] A functionally equivalent signal peptide is one that shares 70% or greater identity with an amino acid sequence, preferably 75% or greater identity, more preferably 80% or greater identity and most preferably 90% or greater identity, such as 95% identity or more, and which retains the ability to secrete the target polypeptide from a prokaryotic cell.

[0018] In many embodiments, DNA sequences which are operably linked are contiguous and, in the case of a secretion leader, contiguous and in the same reading frame.

[0019] Preferably, the linkage between the secretion leader sequence and the polynucleotide encoding the target polypeptide is such that the signal peptide sequence is attached to the N-terminal of the target polypeptide. In certain embodiments, the target polypeptide comprises an N-terminal tag, the linkage between the secretion leader sequence and the polynucleotide encoding the target polypeptide being such that the signal peptide sequence being attached to the tag, preferably to the N-terminus of the tag.

[0020] Polynucleotides comprising a nucleotide sequence encoding a signal peptide with amino acid sequences (SEQ ID NO: 1), (SEQ ID NO: 2), (SEQ ID NO: 3), (SEQ ID NO: 4) or (SEQ ID NO: 5), or a functional equivalent thereof, operably linked to a nucleotide sequence encoding a recombinant polypeptide form a further aspect of the present invention.

[0021] The eukaryotic secretion leader sequence is preferably attached at the 5' end of the polynucleotide encoding the target polypeptide. The nucleotide encoding signal peptide a) preferably has the sequence CATATGCTGAAACGTTCTTCTTGGCTGG CAACTCTGGGTCTGCTGACTGTTGCATCCGTAAGCACTATTGTGTATGCA (SEQ ID NO: 6). The nucleotide encoding signal peptide b) preferably has the sequence CATATGAAGAAAGCTACGTTTATTACTTGCCTGCTGGCTGTTCTGCTGGTTTCTAACC CGATCGTTGTTAACGCG (SEQ ID NO: 7). The nucleotide encoding signal peptide c) preferably has the sequence CATATGAAAGTGTCTGCGGCCGCACTGGCA GTAATCCTGATCGCAACTGCGCTGTGCGCGCCAGCCAGCGCA (SEQ ID NO: 8). The nucleotide encoding signal peptide d) preferably has the sequence CATATGAAAGTTTCTACTGCATTTCTGTGTCTGCTGCTGACTGTTAGCGCATTCTCCG CTCAGGTCCTGGCC (SEQ ID NO: 9). The nucleotide encoding signal peptide e) preferably has the sequence CATATGAAATGTCTGCTGCTGGCGCTGGGTCTGGC ACTGGCATGTGCGGCACAGGCG (SEQ ID NO: 10).

[0022] Promoters which may be employed in the vectors according to the present invention comprise constitutive or inducible promoters. In many preferred embodiments, the promoter is a prokaryotic promoter. Examples of prokaryotic promoters that can be employed include: [0023] a) phage RNA polymerase-dependent promoters, particularly T7 RNA polymerase-dependent promoter systems, preferably single T7 promoters, including those disclosed by Studier and Moffat, J. Mol. Biol. 189:113-130 (1986), incorporated herein by reference, especially a T7 gene 10 promoter; and [0024] b) host RNA polymerase-based promoter systems, especially E. coli RNA polymerase-based promoter systems.

[0025] Examples of preferred promoters which can be employed include T7 gene 10 promoter, T7A1, T7A2, T7A3, .lamda.pL, .lamda.pR, lac, lacUV5, trp, tac, trc, phoA and rrnB.

[0026] When a T7 RNA-polymerase dependent promoter system is employed, it will be recognised that a source of T7 RNA polymerase is required, which is provided by methods known in the art, and commonly by inserting a .lamda.DE3 prophage expressing the required phage polymerase into the host strain to create lysogenic host strains. The T7 RNA polymerase can also be delivered to the cell by infection with a specialised .lamda. transducing phage that carries the gene for the T7 RNA polymerase.

[0027] Operator sequences which may be employed in the expression vector according to the present invention include lac, gal, deo and gln. One or more perfect palindrome operator sequences may be employed. In many preferred embodiments, two perfect palindrome operator sequences are employed, most advantageously one operator sequence being located downstream of the promoter, and one operator sequence being located upstream of the promoter. When two operator systems are employed, the operator sequences are preferably spaced to maximise control of the promoter. In many embodiments, the spacing is from 85 to 150 base pairs apart, preferably from 90 to 126 base pairs apart, and most preferably 91 or 92 base pairs apart. In certain embodiments, an operator sequence overlaps with the transcriptional start point.

[0028] It will be recognised that the operator system is commonly employed with an appropriate repressor sequence. Repressor sequences produce repressor protein, for example lacl gene sequence when using the lac operators. Other lac repressor sequences may also be used, for example the lacl.sup.Q sequence can be used to increase the level of lac repressor protein. The repressor sequence may also be provided by the host cell genome or by using an additional compatible plasmid.

[0029] The expression vector may be integrated into the host cell genome, but is preferably comprised within an extrachromosomal element such as a plasmid. Alternatively, the expression vector may be incorporated into phage or viral vectors and these used to deliver the expression system into the host cell system. The expression vectors can be assembled by methods known in the art.

[0030] The expression vector, particularly when the vector comprises a plasmid, typically also comprises one or more of the following: a selectable marker, for example a sequence conferring antibiotic resistance, and a cer stability sequence.

[0031] The expression vector of the present invention can be employed to express polypeptides, especially proteins in prokaryotic host cells. Examples of prokaryotic cells include bacterial cells, for example gram-negative bacterial cells, including E. coli, Salmonella typhimurium, Serratia marsescens, Pseudomonas putida and Pseudomonas aeruginosa, and gram-positive bacterial cells including Bacillus subtilis. Preferred host cells are bacteria, particularly enterobacteriacae, preferably E. coli, and especially B or K12 strains thereof.

[0032] The expression vector of the present invention is commonly employed in the form of a plasmid. The plasmids may be autonomously replicating plasmids or integrative plasmids.

[0033] The expression vector of the present invention is advantageously employed for the manufacture of polypeptides, especially recombinant proteins, by culturing recombinant cells.

[0034] Polypeptides which can be expressed by the process of the present invention include therapeutic proteins and peptides, including cytokines, growth factors, antibodies, antibody fragments, immunoglobulin like polypeptides, enzyme, vaccines, peptide hormones, chemokines, receptors, receptor fragments, kinases, phosphatases, isomerases, hydrolyases, transcription factors and fusion polypeptides.

[0035] Antibodies which can be expressed include monoclonal antibodies, polyclonal antibodies and antibody fragments having biological activity, including multivalent and/or multispecific forms of any of the foregoing.

[0036] Naturally occurring antibodies typically comprise four polypeptide chains, two identical heavy (H) chains and two identical light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a variable region (V.sub.H) and a constant region (C.sub.H), the C.sub.H region comprising in its native form three domains, C.sub.H1, C.sub.H2 and C.sub.H3. Each light chain comprises a variable region (V.sub.L) and a constant region comprising one domain, C.sub.L.

[0037] The V.sub.H and V.sub.L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V.sub.H and V.sub.L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0038] Antibody fragments which can be expressed comprise a portion of an intact antibody, said portion having a desired biological activity. Antibody fragments generally include at least one antigen binding site. Examples of antibody fragments include: (i) Fab fragments having V.sub.L, C.sub.L, V.sub.H and C.sub.H1 domains; (ii) Fab derivatives, such as a Fab' fragment having one or more cysteine residues at the C-terminus of the C.sub.H1 domain, that can form bivalent fragments by disulfide bridging between two Fab derivatives; (iii) Fd fragment having V.sub.H and C.sub.H1 domains; (iv) Fd derivatives, such as Fd derivatives having one or more cysteine residues at the C-terminus of the C.sub.H1 domain; (v) Fv fragments having the V.sub.L and V.sub.H domains of a single arm of an antibody; (vi) single chain antibody molecules such as single chain Fv (scFv) antibodies in which the V.sub.L and V.sub.H domains are covalently linked; (vii) V.sub.H or V.sub.L domain polypeptide without constant region domains linked to another variable domain (a V.sub.H or V.sub.L domain polypeptide) that is with or without constant region domains, (e.g., V.sub.H-V.sub.H, V.sub.H-V.sub.L, or V.sub.L-V.sub.L) (viii) domain antibody fragments, such as fragments consisting of a V.sub.H domain, or a V.sub.L domain, and antigen-binding fragments of either V.sub.H or V.sub.L domains, such as isolated CDR regions; (ix) so-called "diabodies" comprising two antigen binding sites, for example a heavy chain variable domain (V.sub.H) connected to a light chain variable domain (V.sub.L), in the same polypeptide chain; and (x) so-called linear antibodies comprising a pair of tandem Fd segments which, together with complementary light chain polypeptides, form a pair of antigen binding regions.

[0039] Preferred antibody fragments that can be prepared are mammalian single variable domain antibodies, being an antibody fragment comprising a folded polypeptide domain which comprises sequences characteristic of immunoglobulin variable domains and which specifically binds an antigen (i.e., dissociation constant of 500 nM or less, such as 400 nM or less, preferably 250 nM or less, and most preferably 100 nM or less), and which binds antigen as a single variable domain; that is, without any complementary variable domain. Single variable domain antibodies include complete antibody variable domains as well as modified variable domains, for example in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains. Preferred single variable domains which can be prepared are selected from the group of V.sub.H and V.sub.L, including Vkappa and Vlambda. Most preferably the single variable domains are human or camelid domains, including humanised camelid domains.

[0040] Where the target polypeptide comprises two or more chains to be secreted, particularly where the target polypeptide is a fragment antibody comprising two or more chains, each of the chains is attached to a secretion leader according to the present invention, and polynucleotides encoding such polypeptides are designed accordingly. The secretion leaders employed may be the same or different.

[0041] Accordingly, the present invention also provides a method for the production of a target polypeptide which comprises expressing a vector according to the first aspect of the present invention in a prokaryotic host cell.

[0042] The expression system is expressed by methods well known in the art for the cells employed. Preferred expression methods include culturing the host cells in growth medium, especially by fermentation, and then recovering the expressed polypeptide. The term "growth medium" refers to a nutrient medium used for growing the host cells. In many embodiments, a nutrient solution is employed. Suitable growth media for given host cells and methods of recovering polypeptides are well known in the art.

[0043] Expression may be induced by the addition of an inducer such as isopropyl-.beta.-D-1-thiogalactopyranoside (IPTG), analogues of IPTG such as isobutyl-C-galactoside (IBCG), lactose or melibiose. Other inducers may be used and are described more fully elsewhere (e.g. see The Operon, eds Miller and Renznikoff (1978)). Inducers may be used individually or in combination.

[0044] Preferably, the signal peptide sequence is attached to the N-terminus of the recombinant polypeptide. In certain embodiments, the recombinant polypeptide comprises an N-terminal tag, the signal peptide sequence being attached to the tag, preferably to the N-terminus of the tag.

[0045] Polypeptides comprising signal peptides with amino acid sequences (SEQ ID NO: 1), (SEQ ID NO: 2), (SEQ ID NO: 3), (SEQ ID NO: 4) or (SEQ ID NO: 5), or a functional equivalent thereof, attached to a recombinant polypeptide form another aspect of the present invention.

[0046] Preferably, the signal peptide sequence is attached to the N-terminus of the target polypeptide. In certain embodiments, the linkage between the secretion leader sequence and the polynucleotide encoding the target polypeptide is such that the target polypeptide comprises an N-terminal tag, the signal peptide sequence being attached to the tag, preferably to the N-terminus of the tag.

[0047] Polypeptides comprising signal peptides with amino acid sequences (SEQ ID NO: 1), (SEQ ID NO: 2), (SEQ ID NO: 3), (SEQ ID NO: 4) or (SEQ ID NO: 5), or a functional equivalent thereof, attached to a recombinant target polypeptide form another aspect of the present invention.

[0048] The present invention is illustrated without limitation by the following examples.

EXAMPLE 1

[0049] TAR1-5-19 is an anti-TNF single domain V.sub.L antibody. The amino acid sequence was obtained from International patent application WO2005/035572.

[0050] Construction of Strains

[0051] Strain 1

[0052] A polynucleotide having the sequence:

TABLE-US-00002 (SEQ ID NO: 11) CATATGCTGAAACGTTCTTCTTGGCTGGCAACTCTGGGTCTGCTGACT GTTGCATCCGTAAGCACTATTGTGTATGCAGACATCCAAATGACCCAG TCCCCTTCTTCTCTGAGCGCGTCTGTGGGTGATCGTGTGACCATCACT TGCCGTGCTTCTCAATCCATCGATTCCTACCTGCACTGGTATCAACAG AAACCAGGCAAGGCGCCGAAACTGCTGATTTACTCCGCGTCTGAGCTG CAGTCTGGTGTGCCGAGCCGTTTCTCTGGCTCTGGTTCCGGTACCGAC TTCACTCTGACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACTTAC TACTGCCAACAAGTCGTGTGGCGTCCGTTTACCTTCGGTCAGGGCACG AAAGTGGAAATTAAACGTTGATGACTCGAG

[0053] (where the underlined nucleotides indicate the polynucleotide encoding for the eukaryotic signal peptide sequence MLKRSSWLATLGLLTVASVSTIVYA (SEQ ID NO: 1)) was prepared as an Ndel/Xhol fragment. This fragment was cloned into vector pAVE011, prepared as described in International patent application WO 2007/088371, using the Nde I and Xho I restriction sites in the vector. Recombinant clones were identified by restriction digest and confirmed by sequencing. One plasmid clone was transformed into E. coli strain W3110. An equal amount of overnight culture was mixed with 40% glycerol and aliquoted into cryovials for storage at -70.degree. C.

[0054] Strain 2

[0055] Strain 2 was prepared by the method for Strain 1, except that the polynucleotide prepared had the sequence:

TABLE-US-00003 (SEQ ID NO: 12) CATATGAAGAAAGCTACGTTTATTACTTGCCTGCTGGCTGTTCTGCTG GTTTCTAACCCGATCGTTGTTAACGCGGATATCCAAATGACCCAGTCC CCGAGCTCCCTGTCTGCCAGCGTTGGTGACCGCGTGACTATCACCTGC CGCGCCAGCCAGTCTATTGATTCCTACCTGCATTGGTATCAGCAGAAA CCGGGCAAAGCGCCGAAACTGCTGATCTATTCCGCCAGCGAGCTGCAG TCTGGCGTTCCGAGCCGCTTCTCTGGTTCTGGCTCTGGTACTGACTTC ACCCTGACCATCTCCTCCCTGCAGCCGGAAGACTTCGCTACCTATTAT TGCCAACAGGTGGTTTGGCGTCCATTCACTTTTGGTCAGGGCACCAAA GTAGAAATCAAACGTTAATAACTCGAG

[0056] where the underlined nucleotides indicate the polynucleotide encoding for the eukaryotic signal peptide sequence MKKATFITCLLAVLLVSNPIVVNA (SEQ ID NO: 2).

[0057] Strain 3

[0058] Strain 3 was prepared by the method for Strain 1, except that the polynucleotide prepared had the sequence:

TABLE-US-00004 (SEQ ID NO: 13) CATATGAAAGTGTCTGCGGCCGCACTGGCAGTAATCCTGATCGCAACT GCGCTGTGCGCGCCAGCCAGCGCAGACATCCAGATGACGCAATCTCCG TCTAGCCTGTCCGCGTCCGTGGGCGATCGCGTAACCATTACCTGCCGC GCATCCCAGTCCATCGACAGCTATCTGCACTGGTATCAGCAGAAACCG GGTAAAGCCCCGAAACTGCTGATCTATTCCGCTAGCGAACTGCAGAGC GGCGTTCCGAGCCGTTTCTCCGGCTCTGGTTCTGGTACTGATTTTACC CTGACCATCAGCTCTCTGCAACCGGAAGATTTTGCAACTTATTATTGT CAGCAGGTGGTTTGGCGTCCGTTTACCTTCGGCCAGGGCACCAAAGTC GAGATCAAACGTTGATGACTCGAG

[0059] where the underlined nucleotides indicate the polynucleotide encoding for the eukaryotic signal peptide sequence MKVSAAALAVILIATALCAPASA (SEQ ID NO: 3).

[0060] Strain 4

[0061] Strain 4 was prepared by the method for Strain 1, except that the polynucleotide prepared had the sequence:

TABLE-US-00005 (SEQ ID NO: 14) CATATGAAACTGCTGCTGCTGTCTGCTCTGCTGGGTTGTCTGGCTACT GCGTATGCCGATATCCAAATGACTCAGTCTCCGTCCTCCCTGTCTGCA AGCGTGGGCGATCGTGTCACTATCACCTGCCGTGCGAGCCAGTCTATC AGACTCTTACCTGCATTGGTACCAGCAAAACCGGGCAAAGCTCCTAAA CTGCTGATCTACTCCGCGTCTGAACTGCAGTCTGGCGTTCCGTCTCGT TTCTCTGGCAGCGGTAGCGGCACTGACTTTACCCTGACCATCTCCTCC CTGCAGCCAGAAGATTTTGCGACTTACTATTGCCAGCAGGTGGTGTGG CGCCCGTTCACCTTCGGTCAGGGCACCAAGGTGGAAATTAAGCGTTGA TAACTCGAG

[0062] where the underlined nucleotides indicate the polynucleotide encoding for the eukaryotic signal peptide sequence MKLLLLSALLGCLATAYA (SEQ ID NO: 15).

[0063] Strain 5

[0064] Strain 5 was prepared by the method for Strain 1, except that the polynucleotide prepared had the sequence:

TABLE-US-00006 (SEQ ID NO: 16) CATATGAAAGTTTCTACTGCATTTCTGTGTCTGCTGCTGACTGTTAGC GCATTCTCCGCTCAGGTCCTGGCCGATATCCAGATGACGCAGTCCCCT GTCCTCTCTGAGCGCCAGCTAGGTGATCGCGTAACCATCACGTGCCGT GCATCTCAGAGCATTGATTCTTATCTGCATTGGTACCAGCAGAAGCCG GGCAAAGCGCCGAAACTGCTGATCTATAGCGCTTCCGAGCTGCAGTCC GGTGTACCGTCTCGTTTTTCCGGTTCTGGCAGCGGTACCGATTTCACC CTGACCATCTCCAGCCTGCAGCCGGAGGATTTCGCGACTTATTACTGC CAGCAGGTTGTCTGGCGTCCGTTCACCTTTGGTCAGGGCACGAAAGTT GAAATCAAACGCTGATAACTCGAG

[0065] where the underlined nucleotides indicate the polynucleotide encoding for the eukaryotic signal peptide sequence MKVSTAFLCLLLTVSAFSAQVLA (SEQ ID NO: 4).

[0066] Strain 6

[0067] Strain 6 was prepared by the method for Strain 1, except that the polynucleotide prepared had the sequence:

TABLE-US-00007 (SEQ ID NO: 17) CATATGAAAGTTTCTGCTGCTCTGCTGTGGCTGCTGCTGATTGCTGCT GCTTTCTCTCCGCAGGGTCTGGCCGATATCCAGATGACTCAGTCCCCA TCTAGCCTGAGCGCGTCTGTGGGCGACCGTGTGACTATCACCTGCCGT GCGAGCCAGTCTATCGACTCCTACCTGCATTGGTATCAGCAGAAACCG GGTAAAGCTCCGAAACTGCTGATTTACTCCGCTTCCGAACTGCAGTCT GGCGTACCATCTCGCTTCTCTGGCAGCGGCTCCGGCACCGACTTTACC CTGACTATCTCCTCTCTGCAGCCGGAGGATTTCGCAACGTATTATTGT CAGCAAGTCGTTTGGCGCCCTTTCACCTTCGGTCAGGGCACCAAAGTG GAGATCAAGCGTTGATAACTCGAG

[0068] where the underlined nucleotides indicate the polynucleotide encoding for the eukaryotic signal peptide sequence MKVSAALLWLLLIAAAFSPQGLA (SEQ ID NO: 18).

[0069] Strain 7

[0070] Strain 7 was prepared by the method for Strain 1, except that the polynucleotide prepared had the sequence:

TABLE-US-00008 (SEQ ID NO: 19) CATATGAAAGCGTTTCCAACCTTCGCACTGCTGTTTCTGGTTCTGCTG TTTTCCGCTCACGTTAGCGATGCTGATATCCAAATGACCCAGAGCCCA AGCTCTCTGTCCGCAAGCGTAGGTGACCGTGTTACGATCACCTGCCGT GCGAGCCAGTCTATCGATTCCTACCTGCACTGGTATCAGCAGAAGCCA GGCAAGGCTCCGAAACTGCTGATCTACTCTGCTTCCGAGCTGCAGTCC GGCGTTCCGTCTCGCTTCTCCGGTTCTGGCTCCGGTACCGACTTCACG CTGACCATCTCTTCTCTGCAGCCGGAAGACTTCGCTACTTACTACTGT CAGCAGGTTGTTTGGCGTCCGTTTACTTTCGGCCAGGGTACCAAAGTA GAAATCAAACGTTAATAACTCGAG

[0071] where the underlined nucleotides indicate the polynucleotide encoding for the eukaryotic signal peptide sequence MKAFPTFALLFLVLLFSAHVSDA (SEQ ID NO: 20).

[0072] Strain 8

[0073] Strain 8 was prepared by the method for Strain 1, except that the polynucleotide prepared had the sequence:

TABLE-US-00009 (SEQ ID NO: 21) CATATGAAATGTCTGCTGCTGGCGCTGGGTCTGGCACTGGCATGTGCG GCACAGGCGGACATCCAGATGACGCAGTCTCCATCTAGCCTGTCTGCT TCCGTTGGCGATCGTGTTACCATCACCTGCCGTGCCAGCCAGTCTATC GATTCTTACCTGCACTGGTATCAGCAGAAACCGGGTAAAGCGCCGAAG CTGCTGATCTATTCTGCCTCCGAGCTGCAGAGCGGTGTGCCGTCTCGC TTCTCTGGCTCTGGTTCTGGTACTGACTTTACGCTGACGATTAGCTCC CTGCAGCCGGAGGACTTCGCGACCTATTACTGCCAGCAGGTTGTATGG CGTCCGTTCACGTTCGGCCAGGGTACCAAAGTTGAAATCAAGCGTTAA TAACTCGAG

[0074] where the underlined nucleotides indicate the polynucleotide encoding for the eukaryotic signal peptide sequence MKCLLLALGLALACAAQA (SEQ ID NO: 5).

[0075] Shake-Flask Evaluation

[0076] 10 .mu.l of the thawed glycerol stock was inoculated into 5 ml Luria Broth (LB, 5 g/L yeast extract, 10 g/L tryptone, and 5 g/L sodium chloride) supplemented with tetracycline (10 .mu.g/ml) and glucose (1 g/L). This was incubated at 37.degree. C. in an orbital shaker for 16 h. 500 .mu.l of this culture was then used to inoculate two 250 ml Erlenmeyer flasks containing 50 ml of Luria Broth (composition as described above). The flasks were incubated at 37.degree. C., at 200 rpm in an orbital shaker. Growth was monitored until OD600=0.5-0.7. At this point one flask was induced with IPTG (isopropyl-..beta..-D-1-thiogalactopyranoside) to a final concentration 0.1 mM whilst the second flask was left un-induced and the incubation continued, under the conditions described above for 22 hours, during which samples were taken for measurement of growth, and accumulation of TAR1-5-19 within the bacterial cells. The accumulation levels of TAR1-5-19 was determined using SimplyBlue stained SDS-PAGE gels of whole cell lysates of the sampled bacteria. The harvested cells were further subjected to osmotic shock cell fractionation to isolate the cellular fraction containing proteins that had partitioned in the soluble E. coli periplasmic fraction and the accumulation level in different fractions determined using SimplyBlue stained SDS-PAGE gels. The OS1 (OS=Osmotic Shock) fraction is the supernatant recovered after washing harvested cells in buffer containing sucrose, the OS2 fraction is the supernatant recovered after washing with a low ionic strength buffer, the `supernatant/growth` medium is the residual cell-free residual growth medium and the `cell pellet` is the cell pellet after osmotic shock fractionation. Correctly secreted target polypeptide is detected in the OS1 and/or the OS2 and/or the supernatant/growth medium fractions.

[0077] FIG. 1 shows shake-flask data for Strain 1. It can be seen that a secreted protein of the expected molecular weight was detected in the osmotic shock fractions. This band was subsequently confirmed to be TAR1-5-19 by N-terminal amino acid sequencing. Low level partitioning of TAR1-5-19 into the growth medium was also evident (Lane 2).

[0078] FIG. 2 shows shake-flask data for Strain 2. It can be seen that a secreted protein of the expected molecular weight was detected in the osmotic shock fractions. This band was subsequently confirmed to be TAR1-5-19 by N terminal amino acid sequencing.

[0079] FIG. 3 shows shake-flask data for Strain 3. It can be seen that a secreted protein of the expected molecular weight was detected in the osmotic shock fractions.

[0080] Strain 4 did not accumulate any secreted protein detectable using SimplyBlue stained SDS-PAGE gels.

[0081] FIG. 4 shows shake-flask data for Strain 5 and Strain 6. It can be seen that a secreted protein of the expected molecular weight was detected in the osmotic shock fractions for Strain 5 (according to the present invention), but not Strain 6.

[0082] FIG. 5 shows shake-flask data for Strain 7 and Strain 8. It can be seen that a secreted protein of the expected molecular weight was detected in the osmotic shock fractions, and the growth medium, for Strain 8 (according to the present invention), but not Strain 7.

[0083] Fermenter Evaluation of Strains 1 and 2

[0084] Fermentation inocula for each of Strains 1 and 2 were raised by adding 200 .mu.l of glycerol stock to a 2.0 L baffled shake flask containing 200 mL of Luria Broth (LB) containing 5 g/L yeast extract, 10 g/L peptone, 10 g/L sodium chloride, 10 g/L glucose and 15 mg/L tetracycline. Inocula were grown for 10 h at 37.degree. C. in a shaker-incubator with an agitation of 200 rpm. 20 ml of shake flask inoculum was used to inoculate a 5 L working volume fermenter containing 4 L of batch growth medium (or 45 ml of shake flask inoculum was used to inoculate a 15 L working volume fermenter containing 9 L of batch growth medium for Strain 3). The fermentation was carried out under the operating conditions described below. Temperature was controlled at 37.degree. C. for the first 7-7.5 hours then reduced to 30.degree. C. over a 2 hour period and controlled at 30.degree. C. for the remainder of the fermentation. pH was controlled at 7.0 by automatic addition of 25% (w/v) ammonium hydroxide. The dissolved oxygen tension (dOT) set point was 30% of air saturation and was controlled by automatic adjustment of the fermenter stirrer speed, from a minimum of 250 rpm up to a maximum of 1500 rpm, and supplementation of oxygen to the inlet gas stream. Airflow to the fermenter vessel was 0.5 v/v/min throughout.

[0085] The composition of the batch growth medium is provided in Table 1.

TABLE-US-00010 TABLE 1 Final concentration Component [g/L], [mg/L] and [ml/L] of purified water (NH.sub.4).sub.2SO.sub.4 10.0 g/L Glycerol 35.0 g/L Yeast extract 20.0 g/L NaH.sub.2PO.sub.4 6.0 g/L KH.sub.2PO.sub.4 3.0 g/L NaCl 0.5 g/L MgSO.sub.4.cndot.7H.sub.2O 0.5 g/L CaCl.sub.2.cndot.2H.sub.2O 30 mg/L Antifoam DF204 0.4 ml/L Tetracycline 15 mg/L FeCl.sub.3.cndot.6H.sub.2O 140 mg/L ZnSO.sub.4.cndot.7H.sub.2O 75 mg/L MnSO.sub.4.cndot.H.sub.2O 26 mg/L Na.sub.2MoO.sub.4.cndot.2H.sub.2O 6 mg/L CuSO.sub.4.cndot.5H.sub.2O 7 mg/L H.sub.3.cndot.BO.sub.3 2 mg/L CoCl.sub.2.cndot.6H.sub.2O 6 mg/L

[0086] The composition of the glycerol/ammonium sulphate feed is provided in Table 2.

TABLE-US-00011 TABLE 2 Amount required Component of Feed [g/L] of purified water Glycerol 714 (NH.sub.4).sub.2SO.sub.4 75

[0087] Fermentations were performed in batch mode until depletion of the carbon source (i.e. glycerol) which occurred ca. 10 h post inoculation and was characterized by a sharp rise in dOT. Fed-batch fermentation was initiated at the point of carbon source exhaustion by the addition of a glycerol/ammonium sulphate feed at a feed rate of 2.6-2.9 g of feed per L of medium per hr. Induction was carried out by addition of IPTG to a final concentration of 0.125 mM once the biomass level in the fermentation reached OD600=45-55. The fed-batch phase was continued for 46 hr post induction. The cells and residual cell free growth medium were then harvested. The harvested cells were further subjected to osmotic shock cell fractionation to isolate the cellular fraction containing proteins that had partitioned in the soluble E. coli periplasmic fraction.

[0088] The accumulation of TAR1-5-19 in the soluble periplasmic extract and residual growth medium was estimated as described above. High level secretion of TAR-5-19 was achieved. FIG. 6 shows the data from Strain 1. It can be seen that TAR1-5-19 is secreted and accumulated in the growth medium (S/N). The titre was estimated to be 400 mg/L culture. FIG. 7 shows the data for Strain 2. It can be seen that TAR1-5-19 is secreted and accumulated in the growth medium (S/N). The titre was estimated to be 2400 mg/L culture. The residual pellet fraction following release of the periplasmic fraction (FIG. 6, Lane 9) indicates the accumulation of TAR1-5-19 with the secretion leader. It will be evident to those skilled in the art that further optimisation of the fermentation and induction conditions would increase the secretion of TAR1-5-19 yet further increasing the titre.

EXAMPLE 2

[0089] The ability of vectors according to the present invention to secrete E. coli thioredoxin was investigated. It has been shown that this protein does not secrete very efficiently using typical sec pathway leader sequences such as phoA, due to its rapid folding in the cytoplasm (JOURNAL OF BACTERIOLOGY, October 2003, p. 5706-5713). We compared the secretion of thioredoxin by the present invention with secretion using dsbA and ompA secretion leaders, both of which have been demonstrated to secrete thioredoxin.

[0090] The following gene was synthesised, consisting of the coding sequence for the leader having SEQ ID NO: 4 attached directly to thioredoxin:

TABLE-US-00012 (SEQ ID NO: 22) CATATGAAAGTTTCTACTGCATTTCTGTGTCTGCTGCTGACTGTTAGC GCATTCTCCGCTCAGGTCCTGGCCAGCGATAAAATTATTCACCTGACT GACGAGAGTTTTGACACGGATGTACTCAAAGCGGACGGGGCGATCCTC GTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCG ATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCA AAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATC CGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCA ACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGAC GCTAACCTGGCGTAACTCGAG

[0091] Construction of Plasmid pAB222 and Strain 9

[0092] pAB222 was prepared by the method given for Strain 1, except that SEQ ID NO: 22 was employed, and transformed into E. coli strain W3110, also by the method for Strain 1, to generate Strain 9. Similar constructs were made as controls, which coded for thioredoxin using the dsbA (Strain 10) and ompA (Strain 11) secretion leader sequences.

[0093] Shake-Flask Evaluation

[0094] Shake flask evaluations of Strains 9, 10 and 11 were carried out by the method given in Example 1, and the results shown in FIG. 8.

[0095] The data shows that Strain 9 secretes more thioredoxin into the periplasm than comparative Strains 10 and 11.

EXAMPLE 3

[0096] The ability of vectors according to the present invention to secrete Human growth hormone (hGH) was investigated. hGH has been shown to be secreted at higher levels with a dsbA leader, which is thought to be an srp dependent secretion leader, compared with ompA (Protein Engineering vol. 16 no. 12 pp. 1131-1138, 2003).

[0097] The following gene was synthesised, consisting of the coding sequence for the leader having SEQ ID NO: 4 attached to hGH coding region.

TABLE-US-00013 (SEQ ID NO: 23) CATATGAAAGTTAGCACCGCGTTTCTGTGCTTGCTGTTGACCGTTTCC GCTTTTAGCGCACAAGTCCTGGCCTTTCCTACGATTCCGCTGTCTCGT CTGTTTGATAATGCGATGCTGCGTGCCCATCGTTTGCACCAACTGGCG TTTGACACTTACCAGGAGTTTGAGGAGGCGTATATCCCGAAAGAGCAG AAGTATAGCTTCCTGCAAAACCCGCAAACCAGCCTGTGCTTCAGCGAG TCTATTCCAACCCCGTCTAACCGTGAAGAAACGCAGCAAAAGTCCAAT TTGGAACTGCTGCGCATTAGCCTGCTGCTGATCCAGAGCTGGCTGGAG CCGGTGCAGTTCCTGCGCAGCGTCTTTGCGAACTCCTTGGTGTACGGC GCAAGCGACAGCAATGTGTACGATCTGCTGAAGGACCTGGAAGAGGGT ATTCAGACGTTGATGGGTCGCCTGGAAGATGGTTCGCCGCGTACCGGC CAAATCTTCAAGCAAACGTATAGCAAGTTCGATACCAATAGCCACAAT GACGACGCTCTGCTGAAAAACTACGGCCTGCTGTATTGCTTCCGCAAA GATATGGACAAAGTCGAAACCTTCCTGCGTATTGTGCAGTGTCGTTCC GTTGAAGGTAGCTGTGGTTTCTAACTCGAG

[0098] Strain 12 was prepared by the method given for Strain 1, except that SEQ ID NO: 23 was employed and transformed into E. coli strain W3110.

[0099] Shake-Flask Evaluation

[0100] Shake flask evaluation of Strain 12 was carried out by the method given in Example 1, and the results shown in FIG. 9.

[0101] The data shows that hGH is successfully secreted by Strain 12. After 22 hours incubation post-induction, hGH secretion into the periplasm and its accumulation in both the osmotic shock fractions (OS1 and OS2) is clearly evident using SimplyBlue stained SDS-PAGE gels.

EXAMPLE 4

[0102] The following gene was synthesised, comprising a D1.3 Fab sequence in which both the light and heavy chains were linked to coding sequences for the leader having SEQ ID NO: 4:

TABLE-US-00014 (SEQ ID NO: 24) CATATGAAAGTGAGCACCGCGTTTCTGTGTCTGCTGTTGACGGTGTCT GCGTTTTCCGCACAAGTCCTGGCGCAAGTTCAACTGCAGGAAAGCGGT CCGGGTCTGGTCGCGCCGAGCCAGAGCTTGAGCATCACCTGCACCGTG TCCGGCTTCAGCCTGACCGGCTATGGTGTGAATTGGGTTCGCCAGCCA CCGGGTAAGGGTCTGGAGTGGTTGGGTATGATTTGGGGTGATGGCAAC ACGGACTATAACAGCGCCCTGAAGAGCCGCCTGAGCATCAGCAAGGAC AATAGCAAATCGCAGGTGTTTCTGAAGATGAATAGCTTGCACACCGAC GATACGGCCCGTTACTATTGTGCACGTGAGCGTGACTATCGTCTGGAT TACTGGGGTCAGGGTACCACCGTTACCGTGAGCAGCGCTTCCACCAAG GGCCCGAGCGTGTTCCCGCTGGCCCCGAGCTCTAAGAGCACGAGCGGC GGTACTGCTGCGCTGGGCTGTCTGGTCAAAGATTACTTCCCGGAACCG GTCACCGTGTCTTGGAACAGCGGCGCACTGACCAGCGGCGTTCATACC CCTGCGGTGCTGCAAAGCTCGGGCCTGTACAGCCTGAGCTCTGTTGTC ACTGTTCCGAGCAGCAGCCTGGGTACGCAGACGTACATTTGCAATGTT AATCACAACCCGTCCAACACGAAAGTCGATAAGAAGGTCGAACCGAAG TCCACCAAAACCCATACCTCCGGTGGTGAGCAAAAACTGATTTCGGAG GAGGACCTGAACTAATAAGTCGACGCTAGCGGATCCAAGGAGACTAGT CATATGAAAGTGAGCACCGCGTTCCTGTGCCTGTTGCTGACGGTCAGC GCCTTCAGCGCTCAAGTTCTGGCGGACATTGAGCTGACTCAGAGCCCA GCGAGCCTGAGCGCCAGCGTCGGTGAAACCGTGACCATTACGTGTCGC GCAAGCGGCAACATTCACAACTACCTGGCATGGTATCAGCAAAAACAA GGCAAAAGCCCTCAACTGCTGGTTTACTATACGACCACCCTGGCGGAT GGCGTTCCGAGCCGTTTCTCTGGTTCCGGCTCCGGCACGCAATACTCC TTGAAGATCAATAGCCTGCAGCCGGAAGCGTTTGGTAGCTACTATTGC CAGCACTTTTGGTCTACCCCGCGTACCTTTGGTGGCGGTACCAAGCTG GAAATCAAACGTACGGTTGCAGCGCCGTCCGTGTTCATCTTTCCGCCG AGCGACGAGCAACTGAAGAGCGGTACTGCCTCTGTGGTGTGCCTGCTG AACAATTTCTACCCGCGTGAAGCGAAGGTTCAGTGGAAAGTCGATAAC GCTTTGCAGTCTGGTAATAGCCAAGAGAGCGTGACCGAGCAGGACAGC AAAGATAGCACCTATTCCCTGAGCAGCACCCTGACGCTGAGCAAGGCG GACTACGAAAAGCATAAGGTTTACGCATGTGAGGTCACGCATCAGGGT CTGAGCTCGCCGGTCACCAAATCGTTCAATCGCGGCGAGTCCTAATAA CTCGAG

[0103] Strain 13 was prepared by the method given for Strain 1, except that SEQ ID NO: 24 was employed.

[0104] Shake-Flask Evaluation

[0105] Shake flask evaluation of Strain 13 was carried out by the method given in Example 1, except that no uninduced flask was employed. The accumulation of biologically active D1.3 Fab in the soluble periplasmic extract and residual growth medium was estimated by determining the binding of D1.3 Fab to lysozyme (antigen) in an ELISA assay by reference to a standard curve prepared with purified active D1.3 Fab. It was estimated that 1.2 .mu.g/ml of active D1.3 was produced in the supernatant of these flasks. This demonstrated that the leader can be used for secretion of two separate polypeptide chains, which are subsequently able to form active material in the periplasm.

EXAMPLE 5

[0106] Preparation of Strain 14

[0107] Plasmid pAB222 (prepared by the method of Example 2) was used as the start point for construction of pAB270. The Pseudomonas savastanoi origin of replication was cloned using Polymerase Chain Reaction from Plasmid pCN60 (ATCC 77101; Nieto C, et al. (1990) Gene 87: 145-149). The primers used were F37a (Sequence: 5' AGATCTACGCTTATGGGTGCCTTTCC (SEQ ID NO: 25)) and B29a (Sequence: 5' AGATCTAATACGCAAACCGCCTCTCC (SEQ ID NO: 26). The PCR product was cloned into TOPO TA pCR2.1 (Invitrogen) and then into pAVE187 by Bgl II digestion from pCR2.1. The resultant plasmid, pAB270, was transformed into Pseudomonas putida NCIMB 12018 via electroporation to generate Strain 14.

[0108] Shake Flask Evaluation 10 .mu.l of the thawed glycerol stock was inoculated into 5 ml Nutrient Broth (NB, Oxoid CM0001) supplemented with tetracycline (10 .mu.g/ml). This was incubated at 29.degree. C. in an orbital shaker for 16 h. 500 .mu.l of this culture was then used to inoculate a 250 ml Erlenmeyer flask containing 50 ml of Nutrient Broth (composition as described above). The flask was incubated at 29.degree. C., at 200 rpm in an orbital shaker. Growth was monitored until OD600=0.5-0.7. At this point the flask was induced with IPTG (isopropyl-..beta..-D-1-thiogalactopyranoside) to a final concentration 0.1 mM and the incubation continued, under the conditions described above for 22 hours, during which samples were taken for measurement of growth, and accumulation of thioredoxin within the bacterial cells. The accumulation levels of thioredoxin was determined using SimplyBlue stained SDS-PAGE gels of whole cell lysates of the sampled bacteria. The harvested cells were further subjected to osmotic shock cell fractionation to isolate the cellular fraction containing proteins that had partitioned in the soluble periplasmic fraction.

[0109] The results are shown in FIG. 10 and demonstrate that thioredoxin was secreted by Strain 14.

Sequence CWU 1

1

26125PRTPorphyra purpurea 1Met Leu Lys Arg Ser Ser Trp Leu Ala Thr Leu Gly Leu Leu Thr Val 1 5 10 15 Ala Ser Val Ser Thr Ile Val Tyr Ala 20 25 224PRTPorphyra purpurea 2Met Lys Lys Ala Thr Phe Ile Thr Cys Leu Leu Ala Val Leu Leu Val 1 5 10 15 Ser Asn Pro Ile Val Val Asn Ala 20 323PRTHomo sapiens 3Met Lys Val Ser Ala Ala Ala Leu Ala Val Ile Leu Ile Ala Thr Ala 1 5 10 15 Leu Cys Ala Pro Ala Ser Ala 20 423PRTCavia porcellus 4Met Lys Val Ser Thr Ala Phe Leu Cys Leu Leu Leu Thr Val Ser Ala 1 5 10 15 Phe Ser Ala Gln Val Leu Ala 20 518PRTbubalus bubalis 5Met Lys Cys Leu Leu Leu Ala Leu Gly Leu Ala Leu Ala Cys Ala Ala 1 5 10 15 Gln Ala 678DNAArtificial SequencePolynucleotide encoding Seq ID No. 1 6catatgctga aacgttcttc ttggctggca actctgggtc tgctgactgt tgcatccgta 60agcactattg tgtatgca 78775DNAArtificial SequencePolynucleotide encoding Seq ID No. 2 7catatgaaga aagctacgtt tattacttgc ctgctggctg ttctgctggt ttctaacccg 60atcgttgtta acgcg 75872DNAArtificial SequencePolynucleotide encoding Seq ID No. 3 8catatgaaag tgtctgcggc cgcactggca gtaatcctga tcgcaactgc gctgtgcgcg 60ccagccagcg ca 72972DNAArtificial SequencePolynucleotide encoding Seq ID No. 4 9catatgaaag tttctactgc atttctgtgt ctgctgctga ctgttagcgc attctccgct 60caggtcctgg cc 721057DNAArtificial SequencePolynucleotide encoding Seq ID No. 5 10catatgaaat gtctgctgct ggcgctgggt ctggcactgg catgtgcggc acaggcg 5711414DNAArtificial SequencePolynucleotide used to prepare Strain 1 11catatgctga aacgttcttc ttggctggca actctgggtc tgctgactgt tgcatccgta 60agcactattg tgtatgcaga catccaaatg acccagtccc cttcttctct gagcgcgtct 120gtgggtgatc gtgtgaccat cacttgccgt gcttctcaat ccatcgattc ctacctgcac 180tggtatcaac agaaaccagg caaggcgccg aaactgctga tttactccgc gtctgagctg 240cagtctggtg tgccgagccg tttctctggc tctggttccg gtaccgactt cactctgacc 300atctcttctc tgcagccgga ggatttcgca acttactact gccaacaagt cgtgtggcgt 360ccgtttacct tcggtcaggg cacgaaagtg gaaattaaac gttgatgact cgag 41412411DNAArtificial SequencePolynucleotide used to prepare Strain 2 12catatgaaga aagctacgtt tattacttgc ctgctggctg ttctgctggt ttctaacccg 60atcgttgtta acgcggatat ccaaatgacc cagtccccga gctccctgtc tgccagcgtt 120ggtgaccgcg tgactatcac ctgccgcgcc agccagtcta ttgattccta cctgcattgg 180tatcagcaga aaccgggcaa agcgccgaaa ctgctgatct attccgccag cgagctgcag 240tctggcgttc cgagccgctt ctctggttct ggctctggta ctgacttcac cctgaccatc 300tcctccctgc agccggaaga cttcgctacc tattattgcc aacaggtggt ttggcgtcca 360ttcacttttg gtcagggcac caaagtagaa atcaaacgtt aataactcga g 41113408DNAArtificial SequencePolynucleotide used to prepare Strain 3 13catatgaaag tgtctgcggc cgcactggca gtaatcctga tcgcaactgc gctgtgcgcg 60ccagccagcg cagacatcca gatgacgcaa tctccgtcta gcctgtccgc gtccgtgggc 120gatcgcgtaa ccattacctg ccgcgcatcc cagtccatcg acagctatct gcactggtat 180cagcagaaac cgggtaaagc cccgaaactg ctgatctatt ccgctagcga actgcagagc 240ggcgttccga gccgtttctc cggctctggt tctggtactg attttaccct gaccatcagc 300tctctgcaac cggaagattt tgcaacttat tattgtcagc aggtggtttg gcgtccgttt 360accttcggcc agggcaccaa agtcgagatc aaacgttgat gactcgag 40814393DNAArtificial SequencePolynucleotide used to prepare Strain 4 14catatgaaac tgctgctgct gtctgctctg ctgggttgtc tggctactgc gtatgccgat 60atccaaatga ctcagtctcc gtcctccctg tctgcaagcg tgggcgatcg tgtcactatc 120acctgccgtg cgagccagtc tatcgactct tacctgcatt ggtaccagca aaaaccgggc 180aaagctccta aactgctgat ctactccgcg tctgaactgc agtctggcgt tccgtctcgt 240ttctctggca gcggtagcgg cactgacttt accctgacca tctcctccct gcagccagaa 300gattttgcga cttactattg ccagcaggtg gtgtggcgcc cgttcacctt cggtcagggc 360accaaggtgg aaattaagcg ttgataactc gag 3931518PRTSalmo salar 15Met Lys Leu Leu Leu Leu Ser Ala Leu Leu Gly Cys Leu Ala Thr Ala 1 5 10 15 Tyr Ala 16408DNAArtificial SequencePolynucleotide used to prepare Strain 5 16catatgaaag tttctactgc atttctgtgt ctgctgctga ctgttagcgc attctccgct 60caggtcctgg ccgatatcca gatgacgcag tccccttcct ctctgagcgc cagcgtaggt 120gatcgcgtaa ccatcacgtg ccgtgcatct cagagcattg attcttatct gcattggtac 180cagcagaagc cgggcaaagc gccgaaactg ctgatctata gcgcttccga gctgcagtcc 240ggtgtaccgt ctcgtttttc cggttctggc agcggtaccg atttcaccct gaccatctcc 300agcctgcagc cggaggattt cgcgacttat tactgccagc aggttgtctg gcgtccgttc 360acctttggtc agggcacgaa agttgaaatc aaacgctgat aactcgag 40817408DNAArtificial SequencePolynucleotide used to prepare Strain 6 17catatgaaag tttctgctgc tctgctgtgg ctgctgctga ttgctgctgc tttctctccg 60cagggtctgg ccgatatcca gatgactcag tccccatcta gcctgagcgc gtctgtgggc 120gaccgtgtga ctatcacctg ccgtgcgagc cagtctatcg actcctacct gcattggtat 180cagcagaaac cgggtaaagc tccgaaactg ctgatttact ccgcttccga actgcagtct 240ggcgtaccat ctcgcttctc tggcagcggc tccggcaccg actttaccct gactatctcc 300tctctgcagc cggaggattt cgcaacgtat tattgtcagc aagtcgtttg gcgccctttc 360accttcggtc agggcaccaa agtggagatc aagcgttgat aactcgag 4081823PRTHomo sapiens 18Met Lys Val Ser Ala Ala Leu Leu Trp Leu Leu Leu Ile Ala Ala Ala 1 5 10 15 Phe Ser Pro Gln Gly Leu Ala 20 19408DNAArtificial SequencePolynucleotide used to prepare Strain 7 19catatgaaag cgtttccaac cttcgcactg ctgtttctgg ttctgctgtt ttccgctcac 60gttagcgatg ctgatatcca aatgacccag agcccaagct ctctgtccgc aagcgtaggt 120gaccgtgtta cgatcacctg ccgtgcgagc cagtctatcg attcctacct gcactggtat 180cagcagaagc caggcaaggc tccgaaactg ctgatctact ctgcttccga gctgcagtcc 240ggcgttccgt ctcgcttctc cggttctggc tccggtaccg acttcacgct gaccatctct 300tctctgcagc cggaagactt cgctacttac tactgtcagc aggttgtttg gcgtccgttt 360actttcggcc agggtaccaa agtagaaatc aaacgttaat aactcgag 4082023PRTXenopus laevis 20Met Lys Ala Phe Pro Thr Phe Ala Leu Leu Phe Leu Val Leu Leu Phe 1 5 10 15 Ser Ala His Val Ser Asp Ala 20 21393DNAArtificial SequencePolynucleotide used to prepare Strain 8 21catatgaaat gtctgctgct ggcgctgggt ctggcactgg catgtgcggc acaggcggac 60atccagatga cgcagtctcc atctagcctg tctgcttccg ttggcgatcg tgttaccatc 120acctgccgtg ccagccagtc tatcgattct tacctgcact ggtatcagca gaaaccgggt 180aaagcgccga agctgctgat ctattctgcc tccgagctgc agagcggtgt gccgtctcgc 240ttctctggct ctggttctgg tactgacttt acgctgacga ttagctccct gcagccggag 300gacttcgcga cctattactg ccagcaggtt gtatggcgtc cgttcacgtt cggccagggt 360accaaagttg aaatcaagcg ttaataactc gag 39322405DNAArtificial SequencePolynucleotide used to prepare Strain 9 22catatgaaag tttctactgc atttctgtgt ctgctgctga ctgttagcgc attctccgct 60caggtcctgg ccagcgataa aattattcac ctgactgacg agagttttga cacggatgta 120ctcaaagcgg acggggcgat cctcgtcgat ttctgggcag agtggtgcgg tccgtgcaaa 180atgatcgccc cgattctgga tgaaatcgct gacgaatatc agggcaaact gaccgttgca 240aaactgaaca tcgatcaaaa ccctggcact gcgccgaaat atggcatccg tggtatcccg 300actctgctgc tgttcaaaaa cggtgaagtg gcggcaacca aagtgggtgc actgtctaaa 360ggtcagttga aagagttcct cgacgctaac ctggcgtaac tcgag 40523654DNAArtificial SequencePolynucleotide used to prepare Strain 12 23catatgaaag ttagcaccgc gtttctgtgc ttgctgttga ccgtttccgc ttttagcgca 60caagtcctgg cctttcctac gattccgctg tctcgtctgt ttgataatgc gatgctgcgt 120gcccatcgtt tgcaccaact ggcgtttgac acttaccagg agtttgagga ggcgtatatc 180ccgaaagagc agaagtatag cttcctgcaa aacccgcaaa ccagcctgtg cttcagcgag 240tctattccaa ccccgtctaa ccgtgaagaa acgcagcaaa agtccaattt ggaactgctg 300cgcattagcc tgctgctgat ccagagctgg ctggagccgg tgcagttcct gcgcagcgtc 360tttgcgaact ccttggtgta cggcgcaagc gacagcaatg tgtacgatct gctgaaggac 420ctggaagagg gtattcagac gttgatgggt cgcctggaag atggttcgcc gcgtaccggc 480caaatcttca agcaaacgta tagcaagttc gataccaata gccacaatga cgacgctctg 540ctgaaaaact acggcctgct gtattgcttc cgcaaagata tggacaaagt cgaaaccttc 600ctgcgtattg tgcagtgtcg ttccgttgaa ggtagctgtg gtttctaact cgag 654241542DNAArtificial SequencePolynucleotide used to prepare Strain 13 24catatgaaag tgagcaccgc gtttctgtgt ctgctgttga cggtgtctgc gttttccgca 60caagtcctgg cgcaagttca actgcaggaa agcggtccgg gtctggtcgc gccgagccag 120agcttgagca tcacctgcac cgtgtccggc ttcagcctga ccggctatgg tgtgaattgg 180gttcgccagc caccgggtaa gggtctggag tggttgggta tgatttgggg tgatggcaac 240acggactata acagcgccct gaagagccgc ctgagcatca gcaaggacaa tagcaaatcg 300caggtgtttc tgaagatgaa tagcttgcac accgacgata cggcccgtta ctattgtgca 360cgtgagcgtg actatcgtct ggattactgg ggtcagggta ccaccgttac cgtgagcagc 420gcttccacca agggcccgag cgtgttcccg ctggccccga gctctaagag cacgagcggc 480ggtactgctg cgctgggctg tctggtcaaa gattacttcc cggaaccggt caccgtgtct 540tggaacagcg gcgcactgac cagcggcgtt catacccctg cggtgctgca aagctcgggc 600ctgtacagcc tgagctctgt tgtcactgtt ccgagcagca gcctgggtac gcagacgtac 660atttgcaatg ttaatcacaa cccgtccaac acgaaagtcg ataagaaggt cgaaccgaag 720tccaccaaaa cccatacctc cggtggtgag caaaaactga tttcggagga ggacctgaac 780taataagtcg acgctagcgg atccaaggag actagtcata tgaaagtgag caccgcgttc 840ctgtgcctgt tgctgacggt cagcgccttc agcgctcaag ttctggcgga cattgagctg 900actcagagcc cagcgagcct gagcgccagc gtcggtgaaa ccgtgaccat tacgtgtcgc 960gcaagcggca acattcacaa ctacctggca tggtatcagc aaaaacaagg caaaagccct 1020caactgctgg tttactatac gaccaccctg gcggatggcg ttccgagccg tttctctggt 1080tccggctccg gcacgcaata ctccttgaag atcaatagcc tgcagccgga agcgtttggt 1140agctactatt gccagcactt ttggtctacc ccgcgtacct ttggtggcgg taccaagctg 1200gaaatcaaac gtacggttgc agcgccgtcc gtgttcatct ttccgccgag cgacgagcaa 1260ctgaagagcg gtactgcctc tgtggtgtgc ctgctgaaca atttctaccc gcgtgaagcg 1320aaggttcagt ggaaagtcga taacgctttg cagtctggta atagccaaga gagcgtgacc 1380gagcaggaca gcaaagatag cacctattcc ctgagcagca ccctgacgct gagcaaggcg 1440gactacgaaa agcataaggt ttacgcatgt gaggtcacgc atcagggtct gagctcgccg 1500gtcaccaaat cgttcaatcg cggcgagtcc taataactcg ag 15422526DNAArtificial SequencePrimer F37a 25agatctacgc ttatgggtgc ctttcc 262626DNAArtificial SequencePrimer B29a 26agatctaata cgcaaaccgc ctctcc 26

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