MXPA97006607A - Methods and compositions for secretion of heterolo polypeptides - Google Patents

Abstract

The present invention relato the unexpected result that signal sequences and mutants with reduced translation force provide essentially complete processing and high expression levels of a polypeptide of interest and comparison with wild-type signal sequences, and that many polypeptides of mammal require a narrow range of translation levels to obtain maximum secretion. A set of signal sequence vectors provides a range of translation powers to optimize the expression of a polypeptide of interest

Description

METHODS AND CCMPOSICTCMEf3 FOR SECRETION DB QTJ é IDOg AMPO DE AI TUVEMCTQW This invention relates to signal sequences for the secretion of heterologous polypeptides from bacteria.

"F- &? CpTPfT" w "" t ^ "« ran-m-arai v TfryTCft RtffACTCM? P.V The secretion of heterologous polypeptides in the periplasmic space of E. coli and other prokaryotes or in their culture media is subjected to various parameters. Typically, reactors for secretion of a polypeptide of interest are engineered to place the DNA encoding a 5 'secretory signal sequence relative to the DNA encoding the polypeptide of interest. Two main recurrent problems prevent the secretion of such polypeptides. First, the signal sequence is often incompletely processed or removed, and secondly, the amount of secreted polypeptide is often low or undetectable. The charms for solving these problems fall into three main areas: trying several different signal sequences, REF: 25568 mutating the amino acid sequence of the signal sequence and altering the secretory pathway within the host bacterium. Many signal sequences are available for the first approach, in order to solve the secretion problems. atson (Nucleic Acids Reaearph 12: 5145-5164 (1984)) describes a compilation of signal sequences. U.S. Patent 4,963,495 describes the expression and secretion of mature eukaryotic protein in the periplasmic space of a host organism using a prokaryotic secretion signal sequence DNA attached at its 3 'end to the 5' end of DNA encoding the mature protein. In particular, the DNA encoding the enterotoxin signals of E. coli, especially STII, is preferred. Chang et al. (Gene 55: 189-196 (1987)) describes the use of the STII signal sequence to secrete hGH in E. COli. Gray et al. (Gene 39: 247-245 (1985)) describes the use of the natural signal sequence of human growth hormone and the use of the alkaline phosphatase promoter of E. coli and a signal sequence for the secretion of growth hormone human in E. coli. Wong et al. (Gene 68: 193-203 (1988)) describes the secretion of insulin-like growth factor-1 (IGF-1) fused to the LamB and OmpF secretion leader sequences in E. coli, and the improvement of processing efficiency of these signal sequences in the presence of a prlA4 mutation.

Fujimoto et al. (Bioteph 8: 77-86 (1988)) describes the use of four different E. coli enterotoxin signal sequences, STI, STII, LT-A and LT-B, for the secretion of human epidermal growth factor (hEGF) in E. coli. Denefle et al. (Gene 85: 499-510 (1989)) describes the use of OmpA and phoA signal peptides for the secretion of mature human interleukin lß. The mutagenesis of the signal sequence, in general, has not been especially useful for resolving secretion problems. For example, Morio a-Fujimoto et al. (. Biol. Chem. 266: 1728-1732 (1991)) describes amino acid changes in the LTA signal sequence that increase the amount of human epidermal growth factor secreted in E. coli. Goldstein et al. (J. Bact. 172: 1225-1231 (1990)) describes the substitution of amino acids in the hydrophobic region of OmpA, produces secretion of nuclease A, but not of TEM ß-lactamase. Matteucci et al. (Biotech 4: 51-55 (1986)) describes mutations in the human growth hormone signal sequence that improve the secretion of hGH. Lehnhardt et al. (J. Biol. Chem. 262: 1716-1719 (1987) describes the effect of deletion mutations on the OmpA signal peptide on the secretion of nuclease A and of ß-lactamase TEM.Finally, attempts to improve heterologous secretion In E. coli, by modulating the host's machinery, up to now they have had an improved success in solving secretion problems, for example van Dijl et al. (Mol. Gen. Genet. 227: 40-48 (1991)) describes the effects of the overproduction of the peptidase I signal (SPase I) of E. coli in the processing of precursors Klein et al.

(Protein Engineer n 5: 511-517 (1992) describes the mutagenesis of the LamB signal sequence which has little effect on the secretion of bovine somatotropin, and the bovine somatotropin secretion properties appear to be determined by the mature protein instead of changes in the signal sequence Perez-Perez et al. (Bio / Technology 12: 179-180 (1994)) describes that by providing an E. coli host with additional copies of prlA4 (secY allele) and secE genes, which encode for the major components of the "translocator", ie, the molecular apparatus that physically moves proteins through the membrane, increases the ratio of mature hIL-6 relative to the precursor from 1.2 to 10.8 US Patent Number 5,232,840 describes sites novel binding sites for ribosome useful in improving the production of proteins in bacteria by an improved and / or more efficient translation US Pat. No. 5,082,783 describes s Improved ecretion of heterologous proteins by hosts such as yeast by using promoters of at most intermediate strength with heterologous DNA secretion signal sequences. European patent application number 84308928.5, filed on December 19, 1984, discloses expression elements of promoter-ribosome binding sites of general utility for expression of high level heterologous genes. The present invention describes an unexpected result that altered translation initiation regions with reduced translational force provide essentially complete processing and high levels of secretion of a polypeptide of interest compared to wild-type signal sequences, and that many polypeptides of mammal require a narrow range of translation levels to obtain maximum secretion. A set of vectors with variant translation initiation regions provide a range of translational forces to optimize the secretion of a polypeptide of interest.

BRIEF DESCRIPTION OF IA TUBHd? One aspect of the invention is a method for optimizing the secretion of a heterologous polypeptide of interest in a cell comprising comparing the expression levels of the polypeptide under the control of a set of nucleic acid variants of a translation start region, in wherein the set of variants represents a range of translational forces, and determining the optimal translational force for the production of mature polypeptide, in which the translational force is less than the translational force of the wild-type translation start region. In a further aspect of the invention, the variants are signal sequence variants, especially variants of the STII signal sequence.

BRIEF, DESCRIPTION DB THE DEESHOS Figure 1 shows the sequence of the phoA promoter, trp and the Shine-Dalgarno regions of STII and the STII signal sequence. Figure 2 is a diagram showing the relevant characteristics of the plasmid pLS33. Figure 3 is a diagram showing the construction of the library, pSTIIBK. Figure 4 is a graph showing comparison of the level of IGF-1 expression, as measured by the amount of IGF-1 detected in culture supernatants, for pLS33, pSTIIBK # 131 and pSTIIC. Experiments 1 to 8 represent measurements taken on 8 separate dates.

Figure 5 is a diagram showing the construction of the pSTIIC plasmid. Figure 6 is a diagram showing the construction of the plasmid pSTIILys. Figure 7 is a diagram showing the construction of the ppho21 plasmid. Figure 8 is a diagram showing the construction of ppho31 plasmid. Figure 9 is a diagram showing the construction of the ppho41 plasmid. Figure 10 is a diagram showing the construction of the ppho51 plasmid. Figure 11 is a diagram showing the relevant features of the pSTIICBK library. Figure 12 is a diagram showing the construction of the pSTBKphoA library. Figure 13 is a graph showing phoA activity in isolates from the pSTBKphoA library. Figure 14 shows the nucleotide sequences of the STII signal sequence variants listed. Figure 15 is a diagram showing the construction of plasmid pNT3PST116. Figure 16 is a diagram showing the construction of plasmid pST116pho.

Figure 17 is a diagram showing the relevant characteristics of the "category A" plasmids used in the examples. Figure 18 is a diagram showing the relevant characteristics of the "category B" plasmids used in the examples. Figure 19 is a photograph of a polypeptide gel stained with Coomassie blue showing the secretion of extracellular domains 1 and 2 of mature ICAM-1 in E. coli under the control of variant STII signal sequences. The TIR of the relative force 9 is provided by the ppho31 STII variant; the TIR of the relative force 3 is provided by the ppho41 STII variant. The precursor and mature forms of the polypeptide are indicated in the figures. Figure 20 is a photograph of a polypeptide gel stained with Coomassie blue showing the secretion of mature NT3 in E. coli under control of the STII variant signal sequences. The TIR of the relative force 9 is provided by the ppho31 STII variant; the TIR of the relative force 7 is provided by the ppho21 STII variant; the TIR of the relative force 3 is provided by the ppho41 STII variant; the TIR of the relative force 1 is provided by the variant pphodl STII. The mature form of the polypeptide is indicated in the figure.

Figure 21 is a photograph of a polypeptide gel stained with Coomassie blue showing the secretion of mature RANTES in E. coli under control of the signal sequences of the STII variant. When read from the left to the right in the figure, the relative strength IRRs 9 are provided by the ppho31 and pSTBKphoA variants # 116 STII; the TIR of the relative force 7 is provided by the ppho21 STII variant; in the IRR of the relative force 4 it is provided by the variant pSTBKphoA # 81 STII; the relative strength IRR 3 is provided by the ppho41 STII variant; the relative strength IRR 2 is provided by the variant pSTBKphoA # 107 STII; the relative strength IRRs 1 are provided by pSTBKphoA # 86 and by the STII pphodl variants. The mature form of the polypeptide is indicated in the figure.

I DETAI JADA.

A. DBFIMICIQNES The "translation initiation region" or TIR, as used herein, refers to a region of RNA (or its encoding DNA) determined by the site and efficiency of translation initiation of a gene of interest. (See, for example, McCarthy et al., Trenda in Genetics 6: 78-85 (1990)).

An IRR for a particular gene may extend beyond the ribosome binding site (rbs) to include the 5 'and 3' sequences relative to the rbs. The rbs are defined to include, in a minimal way, the Shine-Dalgarno region and the start codon, plus the bases between them, but may include the expansion of the protected mRNA of the digestion of ribonuclease by bound ribosomes. Therefore, a TIR may include an untranslated leader or the end of an upstream cistron and thus a traditional stop codon. A "secretion signal sequence" or "signal sequence" as used herein, refers to a sequence present in the amino terminus of a polypeptide that directs its translocation through a membrane. Typically, a precursor polypeptide is processed by breaking the signal sequence to generate a mature polypeptide. The term "translational force", as used herein refers to a measurement of a polypeptide secreted in a control system, wherein one or more variants of a TIR are used to direct the secretion of a polypeptide encoded by a indicator gene, and the results are compared with wild-type IRR or with some other control under the same culture and test conditions. For example, in these experiments, the translational force is measured by the use of alkaline phosphatase as the reporter gene expressed under a basal level control for the phoA promoter, in which the secretion of the phoA polypeptide is directed by variants of the STII signal. The amount of mature alkaline phosphatase present in the host is a measure of the amount of secreted polypeptide, and can be quantified in relation to a negative control. Without being limited to one theory, therefore, the "translational force" as used herein may, for example, include a measure of mRNA stability, ribosome binding efficiency at the ribosome site, and a translocation mode through a membrane. As used herein, "polypeptide" generally refers to peptides and polypeptides having at least about 2 amino acids.

B. ÉTODQfi < ? gwgp ^.? eg The present invention demonstrates that translational force is a critical factor in determining whether many heterologous polypeptides are secreted in significant amounts. Therefore, for a given TIR, a series of amino acid or nucleic acid sequence variants can be generated with a range of translational forces, thereby providing a convenient means by which to adjust this factor for optimal secretion of many different polypeptides. The use of an indicator gene expressed under the control of these variants, such as phoA, provides a method for quantifying the relative translational forces of different translation initiation regions. TIR variants or mutants can be provided in the background of a plasmid vector, whereby a set of plasmids into which a gene of interest can be inserted, and its expression can be measured, so as to establish an optimal range of translational forces for maximum expression of the mature polypeptide. Thus, for example, signal sequences can be used from any prokaryotic or eukaryotic organism. Preferably, the signal sequence is STII, OmpA, phoE, LamB, MBP or phoA. The mutagenesis of the TIR is performed by conventional techniques that result in codon changes which can alter the amino acid sequence, although silent changes in the nucleotide sequence are preferred. Alterations in the IRR may include, for example, alterations in the number or separation of the Shine-Dalgarno sequence, together with alterations in the signal sequence. A preferred method for generating mutant signal sequences is the generation of a "codon library" at the beginning of a coding sequence that does not change the amino acid sequence of the signal sequence (ie, silent changes). This can be done by changing the position of the third nucleotide of each codon; additionally, some amino acids such as leucine, serine and arginine, have first and second multiple positions that can add complexity in the elaboration of the bank. This method of mutagenesis is described in detail in Yansura et al. (METHODS: A Companion to MethoHa in Ensymol 4: 151-158 (1992)). Basically, a DNA fragment encoding the signal sequence at the beginning of the mature polypeptide is synthesized so that the third position (and possibly the first and second, as described above) of each of the first 6 to 12 codons is altered The additional nucleotides downstream of these codons provide a site for the binding of a complementary primer used in making the lower strand. The treatment of the upper coding strand and the lower strand primer with DNA polymerase I (Klenow) will result in a set of DNA duplex fragments containing randomized codons. The primers are designed to contain useful cloning sites that can then be used to insert the DNA fragments into an appropriate vector, thereby allowing amplification of the codon library. Alternative methods include, for example, substitution of all rbs with random nucleotides (Wilson et al., BioTechniques 17: 944-952 (1994)) and the use of phage display libraries (see, for example, Barbas et al., Proc. Nat. Acad. Sci. USA 89: 4457-4461 (1992), Garrard et al., Gene 128: 103-109 (1993)). Typically, the TIR variants will be provided in a plasmid vector with elements suitable for expression of a gene of interest. For example, a typical construct will contain a 5 'promoter relative to the signal sequence, a 3' restriction enzyme recognition site relative to the signal sequence for insertion of a gene of interest or a reporter gene, and a selectable marker , such as a marker of drug resistance, for selection and / or maintenance of the bacteria transformed with the resulting plasmids. Promoters suitable for use with prokaryotic hosts include β-lactamase and lactose promoter system (Chang, et al., Nature 275: 617-624 (1978)).; and Goeddel et al., Nature 281: 544-548 (1979)), the alkaline phosphatase and tryptophan (trp) promoter system (Goeddel, Nucleic Acid Res. 8 (18): 4057-4074 (1980) and EP 36,776 ) and hybrid promoters such as the tac promoter (de Boer et al., Proc. Nati. Acad. Sci. USA. 80: 21-25 (1983).

Promoter sequences suitable for use in yeast hosts include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255 (24): 12073-80 (1980) or other glycolytic enzymes (Hess et al. , -T. Adv. Enzyme Reg. 7: 149-67 (1968)) and Holland Riorhemistry 17: 4900-4907 (1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6. -phosphate isomerase, 3-phosphoglyceratomutase, pyruvate kinase, triosephosphate isomerase, phosphoglucosisomerase and glucokinase. Other yeast promoters, which are inducible promoters that have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocitochrome C, acid phosphatase, degrading enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde -3-phosphate dehydrogenase and enzymes responsible for the use of maltose and galactose. Suitable vectors and promoters for use in yeast expression are further described in Hitzeman et al., EP 73,657A. Advantageously, yeast promoters used with yeast promoters can also be used advantageously. Any indicator gene can be used which can be quantified in some way. Thus, for example, the production of alkaline phosphatase can be quantified as a measure of the secreted level of the product of the gene £ llQ_A. Other examples include, for example, the genes for β-lactamase. Preferably, a set of vectors with a range of translational forces is generated in which the DNA encoding a polypeptide of interest can be inserted. This limited set provides a comparison of secreted levels of polypeptides. The secreted level of polypeptides can be determined, for example, by functional assays for the polypeptide of interest, if available, by radioimmunoassays (RIA), enzyme-linked immunoassays (ELISA), or by PAGE and visualization of the correct molecular weight of the polypeptide of interest. Vectors constructed in this way can be used to transform an appropriate host. Preferably, the host is a prokaryotic host. Most preferably, the host is E. coli. Additional details of the invention can be found in the following examples, which further define the scope of the invention. All references mentioned herein are expressly incorporated by reference in their entirety.

EXAMPLES A. CONSTITUTION OF ÜN All of the plasmids described in this patent application are constructed from the basic backbone of pBR322 (Sutcliffe, Cold Spring Harb Symp Ouant Biol 43: 77-90 (1978)). Although the gene of interest expressed in each case varies, the transcriptional and translational sequences required for the expression of each gene are provided by the phoA promoter and by the Shine-Dalgarno sequence for trp (Chang et al., Gañí-i 55: 189 -196 (1987)). Additionally, in the indicated cases, a second Shine-Dalgarno sequence is also present, the Shine-Dalgarno sequence for STII (Picken et al., Infect Immun 42 (1): 269-275 (1983)). The secretion of the polypeptide is directed by the STII signal sequence or variants thereof (Picken et al., Tnfect Immun 42 (1): 269-275 (1983)). The phoA promoter, the Shine-Dalgarno sequences for trp and STII and the wild-type STII signal sequence are provided in Figure 1.

B CQMSIRP? PS33 ION Plasmid pLS33 is derived from phGH1 (Chang et al., Gene 55: 189-196 (1987)), which is constructed for the expression of des (1, 3) -IGF-I. In plasmid pLS33, the gene encoding this insulin-like growth factor-1 version (altered from the original sequence (Elmblad et al., Third European Consensus on Biotechnology III, Weinheim: Verlag Chemie, pp.287-292 ( 1984)) by the removal of the first three amino acids in the N-terminal part) replaces the gene that codes for human growth hormone. The construction of pLS33 maintains the sequences for the phoA promoter, the Shine-Dalgarno regions for trp and STII, and the wild-type STII signal sequence described for phGH1. However, the 3 'end subsequent to the stop codon for des (1,3) -IGF-1 is altered compared to that described for phGH1. In the case of pLS33, immediately downstream of the termination codon a HindIII restriction site has been placed, followed by the methionine start codon of the tetracycline resistance gene of pBR322.

(Sutcliffe, Cold Spring Harb Symp Ouant EIQI 43: 77-90 (1978)). A diagram of plasmid pLS33 is provided in Figure 2.

A plasmid library containing a variable codon library of the STII signal sequence (pSTIIBK) is constructed to examine and search for improved nucleotide sequences of this signal. The vector fragment for the construction of pSTIIBK is generated by isolating the larger fragment when pLS33 is digested with Xbal and BstEII. This vector fragment contains the sequences coding for the phoA promoter, the Shine-Dalgarno sequence for trp and the amino acids 16-67 of des (1,3) -IGF-I. The coding region for amino acids 3-15 of des (1,3) -IGF-I is provided by isolating the DralII-BstEII fragment (approximately 45 bp) from another IGF-I expression plasmid, pLS33LamB. Variations in the nucleotide sequence for the STII signal are derived from two strands of synthetic DNA listed below: '- GCATGTCTAGAATT ATG AA £ AA £ A? X MU QOH SS CJÜ Ü GCN? Cfl ATG m GJTJJ TC X £ H? IH GCT ACA AAC GCG TAT GCC ACTCT - 3' (SEQ ID NO: l) 3 '- CGA TGT TTG CGC ATA CGG TGAGACACGCCACGACTT -5' (SEQ ID NO: 2) R: A, GY: T, CH; A, T, C N: G, A, T, C These two strands of synthetic DNA are aligned or spliced and treated with DNA polymerase I (Klenow fragment) to form duplex DNA of approximately 101 bp. This duplex DNA is then digested with Xbal and DralII to generate the approximately 82 bp fragment encoding the STII signal sequence with variable codons and the first two amino acids of des (1,3) -IGF-I. Then these fragments are ligated together as shown in Figure 3 to build the library, pSTIIBK.

? B PSTIIBKÉ1231 The plasmid library containing a variable codon library of the STII signal sequence (pSTIIBK) is examined for enhanced growth of transformants and increased secretion of IGF-1. Basically, the plasmids are transformed into the 27C7 host chain (see below) and examined for their increased ability to grow in a medium low in phosphate (see Chang et al., Supra) plus carbenicillin (50 μg / ml) based to DO600 cell density measurements. The candidate colonies are tested to determine increased levels of IGF-1 secretion as follows. The colonies are inoculated in 3-5 ml of LB plus carbenicillin (50 μg / ml) and grown at 37 ° C with shaking for about 5-15 hours. The cultures are diluted 1: 100 in 1-3 ml of low medium in phosphates plus carbenicillin (50 μg / ml) and induced with shaking for 24 hours at 37 ° C. The induced cultures are centrifuged in microcentrifuge tubes for 5 minutes. The supernatants are diluted in IGF RIA diluent and stored at -20 ° C until the assay is performed. The amount of IGF-1 secreted in the medium is measured by radioimmunoassay. The expression level of IGF-1, measured by the amount of IGF-1 detected in culture supernatants, is compared for pLS33, pSTIIBK # 131 and pSTIIC, in Figure 4. Variant # 131 consistently improves IGF expression -1 with respect to the "original" or wild-type STII signal sequence. pSTIIC shows some slight improvement in expression with respect to the wild-type sequence. pSTIIBK # 131 differs from wild-type STII in 12 codons and in the deletion of a Shine-Dalgarno sequence. pSTIIC is constructed as described below as a control plasmid having only one Shine-Dalgarno sequence and three codon changes near the end of the 3 'part of the signal.

E. Q3MSTRDCCIÓW OF pSHIg In pSTIIC, the sequence of STII Shine-Dalgarno from plasmid pLS33. In addition, by incorporating silent mutations near the 3 'end of the STII signal, an Mlul site is produced in pSTIIC. The identical fragments described for the construction of pSTIIBK (the pLS33 vector and a DralII-BstEII fragment of approximately 45 bp from pLS33LamB) are used for the construction of this plasmid. However, the synthetic DNA differs from that described above for the construction of pSTIIBK. For the construction of pSTIIC, the synthetic DNA encoding the signal sequence of STII and the first two amino acids of des (1, 3) -IGF-1 is as follows: '- CTAGAATT ATG AAA AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT 3' - TTAA TAC TTT TTC TTA TAG CGT AAA GAA GAA CGT AGA TAC AAG CAÁ Mlul TTT TCT ATT GCT ACA AAC GCG TAT GCC ACTCT - 3 '(SEQ ID NO: 3) AAA AGA TAA CGA TGT TTG CGC ATA CGG TG - 5' (SEQ ID NO: 4) These fragments are ligated together as illustrated in Figure 5 to construct the plasmid pSTIIC.

F. CQlsyrKUCICN OF pSTIIIga The plasmid pSTIILys contains a STII signal sequence that differs from the signal sequence of pSTIIC only in a nucleotide change at the position of the second codon. This signal sequence is constructed from synthetic DNA and placed in a vector based on pBR322 for the expression of the RANTES polypeptide (Schall et al., Im unol 141 (3): 1018-1025 (1988). Xbal-Mlul vector for this construct is isolated from plasmid pBK131Ran (a derivative of plasmid pSTIIBK # 131 with the gene encoding RANTES substituting the gene coding for des (1,3) -IGF-1.) This vector contains the promoter phoA, the Shine-Dalgarno sequence for trp, the last three amino acids of the STIIC signal sequence and the gene encoding the RANTES polypeptide As illustrated in figure 6, this fragment is then ligated with the following strands of DNA Synthetic to build the pSTIILys plasmid (SEQ ID NO: 3): '- CTAGAATT ATG AAG AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT 3 '-TTAA TTC TTC TTC TAG TAG CGT AAA GAA GAA CGT AGA TAC AAG CA TTT TCT ATT GCT ACA AA - 3' (SEQ ID. NO: 5) AAA AGA TAA CGA TGT TTG CGC-51 (SEQ ID NO: 6) G. caNSTagxxad PLASMIDES OF PHOSEAIASA ALKALINE In order to determine the quantitative TIR value for each of the described STII signal sequences, the alkaline phosphatase gene for E. coli is used as the reporter gene. In each of these constructs, the phoA gene is placed downstream with respect to the phoA promoter, the Shp-Dalgarno sequence of trp and a version of the STII signal sequence. Plasmids ppho21, ppho31, ppho41 and pphodl, contain the signal sequences derived from pSTIIC, pLS33, pSTIIBK # 131 and pSTIILys, respectively. In the case of ppho31, the construction also contains the Shine-Dalgarno STII region.

H. LUNtfi'U 'lON DB pphtál The vector fragment for the construction of ppho21 is produced by digesting pBR322 with EcoRI and BamHl, and isolating the largest fragment. The phoA promoter, the Shp-Dalgarno sequence of trp and the signal sequence STII (amino acids 1-20) are provided by isolation of an approximately 484 bp fragment from pCN131Tsc followed by digestion with EcoRI and Mlul. An identical fragment of approximately 484 bp can also be generated from pSTIIC, a plasmid which has been previously described. The phoA gene fragment (approximately 1430 bp) coding for amino acids 24-450 of alkaline phosphatase is generated from plasmid pb0525 after digestion with Bspl286 and BamHI (Inouye et al., J. Bacteriol 146 (2): 668-675 (1981)). This Bspl286-BamHI fragment also contains 142 bp of SV40 DNA (Fiers et al., Nature 273: 113-120 (1978)) after the stop codon of alkaline phosphatase. Synthetic DNA is used to bind the STII signal sequence to the phoA gene. The sequence of this DNA encoding the last three amino acids of the signal sequence of STII and amino acids 1-23 of alkaline phosphatase is as follows: '-CGCGTATGCCCGGACACCAGAAATGCCTGTTCTGGAAAACCGGGCTGCTCAGGGCGATATTACTG 3 '-ATACGGGCCTGTGGTCTTTACGGACAAGACCTTTTGGCCCGACGAGTCCCGCTATAATGAC CACCCGGCGGTGCT-3' (SEQ ID NO: 7) GTGGGCCGCC-5 '(SEQ ID NO: 8) In order to facilitate the construction of this plasmid, the synthetic DNA is pre-ligated to the fragment EcoRI-MluI of pCN131Tsc. This pre-ligation generates a new fragment of approximately 575 bp. As illustrated in Figure 7, the fragment generated from the pre-ligation is then ligated together with the other fragments described to build ppho21.

I. UU STRÜ ION DB ppho31 The vector fragment for the construction of this plasmid is the identical vector described for ppho21. The phoA promoter, the Shp-Dalgarno sequence of trp, the Shine-Dalgarno sequence of STII and the signal sequence of STII (amino acids 1-20) are generated from pJAL55. The necessary fragment (approximately 496 bp) of pJAL55 is isolated after digestion with EcoRI and Mlul. This EcoRI-MluI fragment only differs from the same region of pLS33 by an engineered site Mlul starting at amino acid 20 of the STII signal sequence (as described for pSTIIC). The last three amino acids of the STII signal sequence and the sequence encoding the phoA gene are provided by digesting the ppho21 plasmid with Mlul and BamHl, and isolating the approximately 1505 bp fragment. These fragments are linked together, as shown in Figure 8 to produce ppho31.

J. UNS RÜCCICB The vector fragment for the construction of this plasmid is the identical vector described for ppho21. The phoA promoter, the Shp-Dalgarno sequence of trp and the STII signal sequence with the sequence codons pSTIIBK # 131 (amino acids 1-20) is provided by isolation of an EcoRI-MluI fragment of approximately 484 bp of pNGF131. An identical fragment can also be generated from pSTIIBK # 131. The last three amino acids of the STII signal sequence and the sequence encoding the phoA gene are provided by digesting the ppho21 plasmid with Mlul and BamHl, and isolating a fragment of approximately 1505 bp. As illustrated in Figure 9, these three fragments are then linked together to build ppho41.

K. atfl'KU JlQN DB pphoSl The vector fragment for the construction of pphodl is generated by digesting the plasmid pLS18 with Xbal-BamHl and isolating the largest fragment. Plasmid pLS18 is a derivative of phGH1 (Chang et al., Gene 55: 189-196 (1987)) and an identical vector may have been generated which has used phGH1 instead of pLS18. This Xbal-BamHl vector contains the phoA promoter and the Shine-Dalgarno trp sequence. The STII signal sequence (amino acids 1-20) with the pSTIILys codons is provided by isolating a fragment of approximately 67 bp generated when pSTIILys are digested with Xbal and Mlul. The last three amino acids of the STII signal sequence and the sequence encoding the phoA gene are provided by digesting the ppho21 plasmid with Mlul and BamHl and isolating the approximately 150.5 bp fragment. In Figure 10 a diagram for the construction of pphodl is provided.

L. LOLStif MlK-iLlLQN DB fPTTTf? Y A second variable codon library of the STII signal sequence, pSTIICBK, is constructed. This second codon library is designated solely to focus the codons closest to the start codon, met of the STII signal sequence. As illustrated in Figure 11, pSTIICBK is a plasmid based on pBR322 that contains the gene encoding the RANTES polypeptide (Schall et al.,? L_ Tmmunol 141 (3): 1018-1025 (1988)) under the control of the phoA promoter and the Shp-Dalgarno sequence of trp.

In this plasmid, the secretion of RANTES is driven by a STII signal sequence codon library derived from the following two strands of synthetic DNA: 5 '- GCATGTCTAGAATT ATG AAR AA £ MI? IH flCU TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCC-3 '(SEQ ID NO: 9) 3' -AGA TAA CGA TGT TTG CGC ATA CGG TGA -5 '(SEQ ID NO: 10) R: A, GY: T, CH: A, T, CN: G, A, T, c These two strands of synthetic DNA are annealed and treated with DNA polymerase I (Fragment Klenow) to form DNA duplex of approximately 86 bp. This duplex DNA is then digested with Xbal and Mlul to generate an approximately 67 bp fragment encoding the first 20 amino acids of the STII signal sequence with variable codons at positions 2-6.

M. aa 'HU LQN DB pgTMtfrhoA To increase the number of available STII signal sequences with different relative TIR forces, a convenient method of examining the codon library of pSTIICBK is required. Plasmid pSTBKphoA is constructed as a solution to this problem. In the pSTBKphoA plasmid, the STII codon library of pSTIICBK is inserted upstream of the phoA gene and downstream of the phoA promoter, and the Shpn-Dalgarno sequence of trp. In this way, the activity of phoA provides a means by which to discriminate or determine the difference between the various versions of the STII signal sequences. The vector fragment of this construct is generated by isolation of the larger fragment when pl31TGF is digested with Xbal and BamHl. An identical vector can also be generated from phGH1 (Chang et al., Gene 55: 189-196 (1987)). This vector contains the phoA promoter and the Shine-Dalgarno trp sequence. The codon library of the STII signal sequence is provided by isolating a fragment of approximately 67 bp generated from pSTIICBK after digestion with Xbal and Mlul. The last three amino acids of the STII signal sequence and the sequence encoding the phoA gene are provided by digesting ppho21 with Mlul and BamHl, and isolating a fragment of approximately 1505 bp. As illustrated in Figure 12, the fragments are then ligated together to construct pSTBKphoA.

N. SrCT.tyyrr? W DB pSTBRbhoA #a ?. ace. ? n? . n * Plasmids pSTBKphoA # 81, 86, 107, 116 are selected from the codon library of pSTBKphoA based on their baseline level of phoA activity (Figure 13). As listed in Figure 14, each has a different nucleotide sequence that codes for the STII signal sequence.

O. r? TMH Pn? Pptfiw tm pan i «r > * > This version of the STII signal sequence, ST116, combines the double sequence of Shine-Dalgarno described by Chang et al. (Gene 55: 189-196 (1987)) with the codons of the STII sequence selected from pSTBKphoA # 116. This signal sequence is initially constructed in a plasmid designed for the secretion of the NT3 pro region (pNT3PST116) and then transferred to a plasmid containing the phoA gene to obtain the relative TIR measurement (pST116pho).

P. CCNS ^ RDCTION DB pMT3PSTllg The vector for this construct is generated by digesting plasmid pLS18 with Xbal and BamHl and isolating the largest fragment. Plasmid pLS18 is a derivative of phGH1 (Chang et al., Gene 55: 189-196 (1987)) and an identical vector could be generated from phGH1. This Xbal-BamHl vector contains the phoA promoter and the Shp-Dalgarno sequence of trp. A fragment (approximately 682 bp) containing the last three amino acids of the signal sequence of STII and the coding region for amino acids 19-138 of proNT3 (Jones et al., Proc Nati Acad Sci 87: 8060-8064 (1990) ) is generated from the pNT3P plasmid after digestion with Mlul and BamHl. Plasmid pNT3P is a plasmid based on pBR322 containing the phoA promoter, the STIIBK version # 131 of the STII signal sequence and the coding region for amino acids 19-138 of proNT3. The synthetic DNA strands listed below provide the sequence for the Shine-Dalgarno sequence of STII and the first 20 amino acids of the STII signal sequence: '-CTAGAGGTTGAGGTGATTTT ATG AAA AAA AAC ATC GCA TTT CTT CTT GCA TCT 3' - TCCAACTCCACTAAAA TAC TTT TTT TTG TAG CGT AAA GAA GAA CGT AGA ATG TTC GTT TTT TCT ATT GCT AA AA - 3 '(SEQ ID NO: 11) TAC AAG CAA AAA AGA TAA CGA TGT TTG CGC - 51 (SEQ ID NO: 12) These fragments are then ligated together as shown in Figure 15 to construct pNT3PSTH6.

Q. CQM3IBPCCIQ DB pSTI 1 fiphn The vector for the construction of this plasmid is the identical vector described for the construction of pNT3PST116. The Shine-Dalgarno sequence of STII and the first 20 amino acids of the STII signal sequence are generated (codons pSTBKphoA # 116) by isolating a fragment of approximately 79 bp from pNT3PST116 subsequent to digestion with Xbal and Mlul. The last three amino acids of the STII signal sequence and the sequence encoding the gene for phoA are isolated from pSTBKphoA # 116 after digestion with Mlul and BamHl (a fragment of approximately 1505 bp). As illustrated in Figure 16, ligation or binding of these three fragments results in the construction of pST116pho.

II. ALKALINE PHOSPHATASE ASSAY In these experiments, TIR constructs altered using the phoA reporter gene are tested to determine their relative translational forces by a modification of the method of Amemuura et al. (J. Bacteriol., 152: 692-701, 1982). Basically, the method used is as follows. Plasmids that exhibit altered sequences, either in the IRR, the Shine-Dalgarno region, the nucleotide sequence between the Shine-Dalgarno region and the start codon of the signal sequence, or the signal sequence itself, and whether they are variants of the amino acid sequence or variants of the nucleotide sequence, they are used to transform E. coli, strain 27C7 (ATCC 55,244) although any phoA "strain of E. coli can be used." Transforming colonies are inoculated in medium. Luria-Bertani (LB) plus carbenicillin (50 μg / ml, Sigma, Ine) The cultures are grown at 37 ° C with shaking for 4-8 h The equivalent of 1 OD600 of each culture is centrifuged, then resuspended in 1 ml of strict AP medium (0.4% glucose, 20 mM NH4C1, 1.6 mM MgSO4, 50 mM KCl, 20 mM NaCl, 120 mM triethanolamine, pH 7.4) plus carbenicillin (50 μg / ml). The mixtures are then placed immediately at -20 ° C overnight. After reheating, a drop of toluene is added to 1 μl of reheated culture. After vortexing, the mixtures are transferred to 16 x 125 mm test tubes and aerated in a wheel at 37 ° C for 1 hour. Then 40 μl of each culture treated with toluene is added to 1 ml of 1M Tris-HCl, pH 8, plus PNPP (disodium 4-nitrophenylphosphate hexahydrate) 1 mM and left at room temperature for 1 hour. The reactions are stopped by adding 100 ml of 1 M sodium phosphate, pH 6.5. The DO410 is measured in the next 30 minutes. The enzymatic activity is calculated as micromoles of p-nitrophenol released per minute, per one equivalent of DO600 cells. The results are summarized in Table 1.

Table 1. Determination of the Relative Strength of the IRR: Use of phoA as an Indicator Gene TIR Deviation Activity Relative Strength phoA1 standard pBR322 0.0279 0.0069 pphodl * 0.0858 0.0165 1 pSTBKp oA # 86 0.1125 0.0246 1 pSTBKphoA # 107 0.1510 0.0267 2 pphc-413 0.1986 0.0556 3 pSTBKphoA # 81 0.2796 0.0813 4 ppho214 0.4174 0.1145 7 pSTBKph? A # 116 0.5314 0.1478 9 ppho315 0.5396 0.0869 9 pST116pho 0.7760 0.1272 13 ^ icromoles of p-nitrophenol / min / cells DO600 2 same variant of STII as pSTIILys 3 same variant of STII as pSTIIBK # 131 4 same variant of STII as STIIC 5 STII wild type + site Mlul, last codon GCC, III. SECRETION OF EXAMPLES OF HETEROLLIC POLYPEPTIDES The plasmids used in these examples are all very similar in design to those described above. Instead of describing each construction in detail, the expression plasmids are described here in general terms. Although different polypeptides of interest are expressed in each example, the only significant variation between these constructs is the nucleotide sequence subsequent to the 3 'end of each coding region. Therefore, for descriptive purposes, these plasmids are generally grouped into the following two categories, based on their 3 'sequence: Category A: Within about 25 bp, 3 'to the stop codon of each gene of interest begins the sequence encoding the transcriptional terminator described by Scholtissek and Grosse (Nucleic Acid Res. 15 (7): 3185 (1987)) followed by the tetracycline resistance gene of pBR322 (Sutcliffe, Cold Spring Harb Sy p Ouant Biol 43: 77-90 (1978)). Examples in this category include plasmids designed for the secretion of mature NGF (Ullrich et al., Nature 303: 821-825 (1983)), TGF-ßl MADURO (Derynck et al., Nature 316: 701-705 (1985)) and domains 1 and 2 of ICAM-1 (Staunton et al., Cell 52: 925-933 (1988). A schematic representation of these plasmids is provided in Figure 17.

Category B_ Examples in this category include plasmids designed for the secretion of mature VEGF (Leung et al., Science 246: 1306-1309 (1989)), mature NT3 (Jones et al., Proc. Nati. Acad. Sci. USA 87: 8060-8064 (1990)), RANTES (Schall et al., J. Immunol 141 (3): 1018-1025 (1988)) and phoA. The termination codon of each of these plasmids is followed in the 3 'direction by a segment of untranslated DNA (VEGF: approximately 43 bp, mature NT3, approximately 134 bp, RANTES: approximately 7 bp, phoA: approximately 142 bp) . After this 3 'untranslated region, the pBR322 sequence is restarted starting with the HindIII site (as in the mature NT3 secretion plasmid) or the BamH1 site (phoA secretion plasmids, VEGF, RANTES). A schematic representation of the plasmids included in this category is illustrated in Figure 18. These plasmids are used to transform the host E. coli, strain 27C7. The transformant colonies are inoculated in 3-5 ml of LB + carbenicillin (50 μg / ml). The cultures are grown at 37 ° C with shaking for 3-8 h. The cultures are then diluted 1: 100 in 3 ml of low phosphate medium (Chang et al., Supra) and induced for approximately 20 hours with shaking at 37 ° C. For each growth culture, an aliquot of DO600 0.5 is centrifuged in a microcentrifuge tube. Then each DO6000.5 pellet is prepared for gel analysis as follows. Each pellet is resuspended in 50 μl of TE (10 mM Tris, pH 7.6, 1 mM EDTA). After the addition of 10 μl of 10% SDS, and 5 μl of reducing agent (1 M dithiothreitol or 1 M β-mercaptoethanol), the samples are heated to about 90 ° C for 2 minutes and then vortexed. The samples are allowed to cool to room temperature, after which 500 μl of acetone is added. The mixtures are vortexed and then allowed to stand at room temperature for about 15 minutes. The mixtures are centrifuged for 5 minutes. Supernatants are discarded, and the pellets are resuspended in 20 μl of water, 5 μl of reducing agent, 25 μl of sample buffer NOVEX 2X. The samples are heated to approximately 90 ° C for 3-5 minutes, and then vortexed. After centrifugation for 5 minutes, the supernatants are transferred to clean tubes and the pellets are discarded. 5-10 μl of each sample is loaded into 10 wells, 1.0 mm of NOVEX manufactured gel (San Diego, CA) and electrophoresed for 1.5-2 hours at 120 volts. The gels are stained with Coomassie blue to visualize the polypeptide (Figures 19-21). To provide additional quantification of the results, some gels are analyzed by densitometry. These results are shown in table 2 below. Both results of polypeptide gels and densitometry indicate that the heterologous polypeptides tested are secreted in a consistently more efficient manner when a STII variant of reduced translational force is used to direct the secretion of that polypeptide.

Table 2. Examples of Enhanced Polypeptide Secretion by Modification of the? R Densitometry Tests of the Polypeptide Gels Polypeptide R (Relative Strength) Secreted Quantity (Total% of the host polypeptide VEGF 9 0.6 3 5.9 NGF 9 1.6 7 1.8 4 5.7 1 5.5 RANTES 9 0.3 9 0.2 7 0.4 4 3.9 3 3.6 2 3.5 1 * 1.6 1 1.7 GF-ßl 7 1.7 3 9.2 signal awareness * pSTBKphoA # il? SEQUENCE LIST t (1. GENERAL INFORMATION: (i) APPLICANT: Genentech, Inc. (ii) TITLE OF THE INVENTION: Methods and compositions for the secretion of heterologous polypeptides (iii) NUMBER OF SEQUENCES: 23 . { iv) CORRESPONDENCE ADDRESS: (A) RECIPIENT: Genentech, Inc. (B) STREET: 460 Point San Bruno Blvd (C) CITY: South San Francisco (D) STATE: California (E) COUNTRY: USA (F) AREA POSTCARD: 94080 (v) READABLE FORM OF THE COMPUTER: (A) TYPE OF MEDIUM: 3.5 inches, 1.44 Mb flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: WinPatin (Genentech) (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATA: (A) NUMBER OF APPLICATION: 08/398615 (B) DATE OF SUBMISSION: 01-MARCH-1995 (viii) ATTORNEY / AGENT INFORMATION: (A) NAME: Lee, Wendy M. (B) REGISTRATION NUMBER: 00,000 (C) REFERENCE NUMBER / FILE: P0889 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 415 / 225-1994 (B) TELEFAX: 415 / 952-9881 (C) TÉLEX: 910 / 371-7168 (2) INFORMATION FOR SEC. FROM IDENT. NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 88 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: l: GCATGTCTAG AATTATGAAR AARAAYATHG CNTTYCTNCT NGCNTCNATG 50 TTYGTNTTYT CNATHGCTAC AAACGCGTAT GCCACTCT 88 (2) INFORMATION FOR SEC. FROM IDENT. NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 2: TTCAGCACCG CACAGAGTGG CATACGCGTT TGTAGC 36 (2) INFORMATION FOR SEC. FROM IDENT. NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 82 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 3: CTAGAATTAT GAAAAAGAAT ATCGCATTTC TTCTTGCATC TATGTTCGTT 50 TTTTCTATTG CTACAAACGC GTATGCCACT CT 82 (2) INFORMATION FOR SEC. FROM IDENT. NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 75 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 4: GTGGCATACG CGTTTGTAGC AATAGAAAAA ACGAACATAG ATGCAAGAAG 50 AAATGCGATA TTCTTTTTCA TAATT 75 (2) INFORMATION FOR SEC. FROM IDENT. NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 67 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 5: CTAGAATTAT GAAGAAGAAT ATCGCATTTC TTCTTGCATC TATGTTCGTT 50 TTTTCTATTG CTACAAA 67 (2) INFORMATION FOR SEC. FROM IDENT. NO 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 67 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO 6: CGCGTTTGTA GCAATAGAAA AAACGAACAT AGATGCAAGA AGAAATGCGA 50 TATTCTTCTT CATAATT 67 (2) INFORMATION FOR SEC. FROM IDENT. NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 79 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 7: CGCGTATGCC CGGACACCAG AAATGCCTGT TCTGGAAAAC CGGGCTGCTC 50 AGGGCGATAT TACTGCACCC GGCGGTGCT 79 (2) INFORMATION FOR SEC. FROM IDENT. NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 71 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 8: CCGCCGGGTG CAGTAATATC GCCCTGAGCA GCCCGGTTTT CCAGAACAGG 50 CATTTCTGGT GTCCGGGCAT A 71 (2) INFORMATION FOR SEC. FROM IDENT. NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 83 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 9: GCATGTCTAG AATTATGAAR AARAAYATHG CNTTTCTTCT TGCATCTATG 50 TTCGTTTTTT CTATTGCTAC AAACGCGTAT GCC 83 (2) INFORMATION FOR SEC. FROM IDENT. NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 10: AGTGGCATAC GCGTTTGTAG CAATAGA 27 (2) INFORMATION FOR SEC. FROM IDENT. NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 79 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 11: CTAGAGGTTG AGGTGATTTT ATGAAAAAAA ACATCGCATT TCTTCTTGCA 50 TCTATGTTCG TTTTTTCTAT TGCTACAAA 79 (2) INFORMATION FOR SEC. FROM IDENT. NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 79 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 12: CGCGTTTGTA GCAATAGAAA AAACGAACAT AGATGCAAGA AGAAATGCGA 50 TGTTTTTTTTT CATAAAATCA CCTCAACCT 79 (2) INFORMATION FOR SEC. FROM IDENT. NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 506 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 13: GAATTCAACT TCTCCATACT TTGGATAAGG AAATACAGAC ATGAAAAATC 50 TCATTGCTGA GTTGTTATTT AAGCTTGCCC AAAAAGAAGA AGAGTCGAAT 100 GAACTGTGTG CGCAGGTAGA AGCTTTGGAG ATTATCGTCA CTGCAATGCT 150 TCGCAATATG GCGCAAAATG ACCAACAGCG GTTGATTGAT CAGGTAGAGG 200 GGGCGCTGTA CGAGGTAAAG CCCGATGCCA GCATTCCTGA CGACGATACG 250 GAGCTGCTGC GCGATTACGT AAAGAAGTTA TTGAAGCATC CTCGTCAGTA 300 AAAAGTTAAT CTTTTCAACA GCTGTCATAA AGTTGTCACG GCCGAGACTT 350 ATAGTCGCTT TGTTTTTATT TTTTAATGTA TTTGTAACTA GTACGCAAGT 400 TCACGTAAAA AGGGTATCTA GAGGTTGAGG TGATTTTATG AAAAAGAATA 450 TCGCATTTCT TCTTGCATCT ATGTTCGTTT TTTCTATTGC TACAAATGCC 500 TATGCA 506 (2) INFORMATION FOR SEC. FROM IDENT. NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 amino acids (B) TYPE: Nucleic acid (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 14: Met Lys Lys Asn lie Wing Phe Leu Leu Wing Being Met Phe Val Phe 1 5 10 15 Ser lie Ala Thr Asn Ala Tyr Ala 20 23 (2) INFORMATION FOR SEC. FROM IDENT. NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 90 base pairs (B> TYPE: Nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 15: TCTAGAGGTT GAGGTGATTT TATGAAAAAG AATATCGCAT TTCTTCTTGC 50 ATCTATGTTC GTTTTTTTCTA TTGCTACAAA YGCSTATGCM 90 (2) INFORMATION FOR SEC. FROM IDENT. NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 78 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 16: TCTAGAATTA TGAAAAAGAA TATCGCATTT CTTCTTGCAT CTATGTTCGT 50 TTTTTCTATT GCTACAAACG CGTATGCM 78 (2) INFORMATION FOR SEC. FROM IDENT. NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 78 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 17: TCTAGAATTA TGAAGAAGAA TATTGCGTTC CTACTTGCCT CTATGTTTGT 50 CTTTTCTATA GCTACAAACG CGTATGCM 78 (2) INFORMATION FOR SEC. FROM IDENT. NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 78 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 18: TCTAGAATTA TGAAGAAGAA TATCGCATTT CTTCTTGCAT CTATGTTCGT 50 TTTTTCTATT GCTACAAACG CGTATGCM 78 (2) INFORMATION FOR SEC. FROM IDENT. NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 78 base pairs (B) TYPE: Nucleic acid (OR TYPE OF HEBRA: Double (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 19: TCTAGAATTA TGAAAAAAAA CATCGCATTT CTTCTTGCAT CTATGTTCGT 50 TTTTTCTATT GCTACAAACG CGTATGCM 78 (2) INFORMATION FOR SEC. FROM IDENT. NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 78 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 20: TCTAGAATTA TGAAAAAAAA CATTGCCTTT CTTCTTGCAT CTATGTTCGT 50 TTTTTCTATT GCTACAAACG CGTATGCM 78 (2) INFORMATION FOR SEC. FROM IDENT. NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 78 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 21: TCTAGAATTA TGAAGAAAAA CATCGCTTTT CTTCTTGCAT CTATGTTCGT 50 TTTTTCTATT GCTACAAACG CGTATGCM 78 (2) INFORMATION FOR SEC. FROM IDENT. NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 78 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 22: TCTAGAATTA TGAAAAAGAA CATAGCGTTT CTTCTTGCAT CTATGTTCGT 50 TTTTTCTATT GCTACAAACG CGTATGCM 78 (2) INFORMATION FOR SEC. FROM IDENT. NO: 23 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 90 base pairs (B) TYPE: Nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT.

TCTAGAGGTT GAGGTGATTT TATGAAAAAA AACATCGCAT TTCTTCTTGC 50 ATCTATGTTC GTTTTTTTCTA TTGCTACAAA CGCGTATGCM 90 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (21)

RJBIVTMDICACTQNES

1. A method for optimizing the secretion of a heterologous polypeptide of interest in a cell, characterized in that it comprises comparing the expression levels of the polypeptide under the control of a set of nucleic acid variants of a translation initiation region, wherein the of variants represents a range of translational forces, and determining the optimal translation force for the production of a mature polypeptide, in which the optimal translational force is less than the translational force of the wild-type translation start region.

2. A method for secreting a heterologous polypeptide of interest in a cell, characterized in that it comprises using a variant of the translation initiation region operably linked to nucleic acid encoding the heterologous polypeptide, to secrete the heterologous polypeptide, wherein the The translational strength of the variant translation initiation region is less than the translational strength of the wild type translational initiation region.

3. The method according to claim 1 or 2, characterized in that the variant does not alter the amino acid sequence of the translational start region.

4. The method according to claim 1 or 2, characterized in that the amount of polypeptide secreted when the nucleic acid is operably linked to the variant is greater than the amount of secreted polypeptide when the nucleic acid is operably linked to the region start of wild type translation.

5. The method according to claim 1 or 2, characterized in that the variants comprise nucleic acid variants of a secretion signal sequence.

6. The method according to claim 5, characterized in that the secretion signal sequence is a prokaryotic secretion signal sequence.

7. The method according to claim 6, characterized in that the variant secretion signal sequences are variants of STII, OmpA, PhoE, LamB, MBP or PhoA.

8. The method according to claim 7, characterized in that the variant signal sequence is a variant of STII.

9. The method according to claim 8, characterized in that the variant is selected from the group consisting of: 5 'TCTAGAGGTTGAGGTGATTTT ATG AAA AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ATA GCS TAT GCM 3' (SEQ ID NO: 15); 5 * TCTAGAATT ATG AAA AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT HERE AAC GCG TAT GCM 3 '(SEQ ID NO: 16); 5 'TCTAGAATT ATG AAG AAG AAT ATT GCG TTC CTA CTT GCC TCT ATG TTT GTC TTT TCT ATA GCT ACA AAC GCG TAT GCM 3' (SEQ ID NO: 17); 5 'TCTAGAATT ATG AAG AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3' (SEQ ID NO: 18); 5 'TCTAGAATT ATG AAA AAA AAC ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3' (SEQ ID NO: 19); 5 • TCTAGAATT ATG AAA AAA AAC ATT GCC TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 31 (SEQ ID NO: 20); 5 'TCTAGAATT ATG AAG AAA AAC ATC GCT TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3 * (SEQ ID NO: 21); 5 'TCTAGAATT ATG AAA AAG AAC ATA GCG TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3"(SEQ ID NO: 22); 5 'TCTAGAGGTTGAGGTGATTTT ATG AAA AAA AAC ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3' (SEQ ID NO: 23).

10. A nucleic acid encoding a variant of translational start region, characterized in that the translational force of the variational translational start region is less than the translational strength of the wild-type translational start region.

11. A nucleic acid encoding a polypeptide operably linked to a translational start region variant, characterized in that the translational force of the variational translational start region is less than the translational strength of the wild-type translational start region.

12. The nucleic acid according to claim 10 or 11, characterized in that the variant does not alter the amino acid sequence of the translational start region.

13. The nucleic acid according to claim 10 or 11, characterized in that the amount of polypeptide secreted when the nucleic acid is operably linked to the variant is greater than the amount of secreted polypeptide when the nucleic acid is operably linked to the nucleic acid. wild type translation start region.

14. The nucleic acid according to claim 10 or 11, characterized in that the variants comprise nucleic acid variants of a secretion signal sequence.

15. The nucleic acid according to claim 10 or 11, characterized in that the secretion signal sequence is a prokaryotic secretion signal sequence.

16. The nucleic acid according to claim 15, characterized in that the variant secretion signal sequences are variants of STII, OmpA, PhoE, LamB, MBP or PhoA.

17. The nucleic acid according to claim 16, characterized in that the variant secretion signal sequence is a variant of STII.

18. The nucleic acid according to claim 17, characterized in that the variant is selected from the group consisting of: 5 'TCTAGAGGTTGAGGTGATTTT ATG AAA AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ATA GCS TAT GCM 3 * (SEQ ID NO: 15); 5 'TCTAGAATT ATG AAA AAG AAT ATC GCA TTT CTT CTT OCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3' (SEQ ID NO: 16); 5 'TCTAGAATT ATG AAG AAG AAT ATT GCG TTC CTA CTT GCC TCT ATG TTT GTC TTT TCT ATA GCT ACA AAC GCG TAT GCM 3' (SEQ ID NO: 17); 5 'TCTAGAATT ATG AAG AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3' (SEQ ID NO: 18); 5 'TCTAGAATT ATG AAA AAA AAC ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3' (SEQ ID NO: 19); 5 'TCTAGAATT ATG AAA AAA AAC ATT GCC TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3' (SEQ ID NO: 20); 5 • TCTAGAATT ATG AAG AAA AAC ATC GCT TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3 '(SEQ ID NO: 21); 5 'TCTAGAATT ATG AAA AAG AAC ATA GCG TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3' (SEQ ID NO: 22); Y 5"TCTAGAGGTTGAGGTGATTTT ATG AAA AAA AAC ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT HERE AAC GCG TAT GCM 3 • (SEQ ID NO: 23).

19. An expression vector characterized in that it comprises the nucleic acid according to any of claims 10 to 18, operably linked to additional elements for expression for a gene of interest.

20. A host cell, characterized in that it comprises the nucleic acid according to any of claims 10 to 19.

21. A variant of the STII secretion signal sequence characterized in that it has the nucleic acid sequence consisting of: 5 'TCTAGAGGTTGAGGTGATTTT ATG AAA AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ATA GCS TAT GCM 3' (SEQ ID NO: 15); 5 'TCTAGAATT ATG AAA AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3' (SEQ ID NO: 16); 5 'TCTAGAATT ATG AAG AAG AAT ATT GCG TTC CTA CTT GCC TCT ATG TTT GTC TTT TCT ATA GCT ACA AAC GCG TAT GCM 3' (SEQ ID NO: 17); 5 'TCTAGAATT ATG AAG AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3' (SEQ ID NO: 18); 5 'TCTAGAATT ATG AAA AAA AAC ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3' (SEQ ID NO: 19); 5 'TCTAGAATT ATG AAA AAA AAC ATT GCC TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3' (SEQ ID NO: 20); 5 'TCTAGAATT ATG AAG AAA AAC ATC GCT TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3' (SEQ ID NO: 21); 5 »TCTAGAATT ATG AAA AAG AAC ATA GCG TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3 '(SEQ ID NO: 22); Y 5 'TCTAGAGGTTGAGGTGATTTT ATG AAA AAA AAC ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAT GCM 3' (SEQ ID NO: 23).