CN100564540C - Make up the leading peptide of secreting bacteria recombinant protein - Google Patents

Abstract

The invention provides the method for separating leading peptide, leading Toplink guidance of heterologous protein matter is exported from bacterial cytoplasm.Therefore this method depends on the sequence that can export rapidly from the library screening of inferring leading peptide or leading peptide mutant, can protect in the tenuigenin short-lived reporter protein not degraded.This paper mutant leader peptides identified shows provides significantly higher output of steady-state level, not only is used for short-lived reporter protein, also is used for other stable long-acting protein.These leading peptides can be used for instructing or improving protein secreting.The present invention further discloses through the folding protein of Tat approach output tenuigenin.The protein of tool disulfide linkage is at first folding in cytoplasm in suitable oxidizing mutant strains.The protein of this tenuigenin pre-folded contains disulfide linkage, with after Tat approach output.

Description

Make up the leading peptide of secreting bacteria recombinant protein

Background of invention

The U.S. Provisional Application the 60/337th that the application's requirement is submitted to November 5 calendar year 2001 with the name of George Georgiou and Matthew DeLisa, the U.S. Provisional Application the 60/404th that No. 452 and on August 21st, 2002 submit to, No. 994 right of priority, described application are entitled as " leading peptide that makes up the secreting bacteria recombinant protein ".The spy incorporates these two applications into for your guidance in full at this.

1. background of invention

The present invention relates generally to genetic engineering and protein secreting field.Particularly, the application relates to the leading peptide of secreting bacteria recombinant protein.

2. description of related art

From tenuigenin excretory protein is generally to be about 15-30 amino acid whose N-terminal peptide extension synthetic with what be called leading peptide.Can be outside outputing to kytoplasm remove this leading peptide by proteolysis from sophisticated protein during the position or after this.

In fact nearest discovery is presented at four kinds of protein output pathways (Stauart and Neupert, 2000) in the Gram-negative bacteria: general (Sec) approach (Danese and Silhavy, 1998 of secreting; Pugsley, 1993), signal recognition particle (SRP) dependence approach (Meyer etc., 1982), YidC rely on approach (Samuelson etc., 2000) and double arginine transposition (Tat) system (Berks, 1996).Plant in these approach at first three, polypeptide warp ' screw thread ' (threading) mechanism is striden film, promptly folding polypeptide inserts the hole-similar structures of Protein S ecY, SecE and SecG formation and strides film (Schatz and Dobberstein, 1996) through the process promotion of need hydrolysising ATP.

On the contrary, with part or perhaps even fully folding conformation transmembrane through the protein of Tat approach output.' Δ pH dependence ' the protein import pathway closely mutually relevant (Settles etc., 1997) of bacterium Tat system and plant chloroplast thylakoid membrane.Do not need the ATP hydrolysis and do not comprise passing through through the output of Tat approach through the SecY/E/G hole.In most of situation, the natural substrate of this approach is a protein, and they need in tenuigenin folding to obtain cofactor such as the FeS center or the molybdopterin of certain limit.Yet, do not contain cofactor but folding too quickly or tension and can not can from tenuigenin, secrete (Berks, 1996 by being fused to the special leading peptide of Tat-through the protein of any other approach output; Berks etc., 2000).

Membranin TatA, TatB and TatC are neccessary composition (Sargent etc., 1998 of Tat transposase in the intestinal bacteria; Weiner etc., 1998).In addition, although TatA homologous TatE is unessential, also can in transposition, works and can not get rid of the participation of other factor.TatA, TatB and TatE are conformity membrane albumen, estimate with them towards cytoplasmic C-end structure territory across inner membrance once.Prediction TatA and TatB albumen be single across albumen, and TatC albumen has 6 to stride membrane portions and estimate as easily bit port and preceding protein receptor performance function (Berks etc., 2000; Bogsch etc., 1998; Chanal etc., 1998).Mutation T atB or C remove output (Bogsch etc., 1998 fully; Sargent etc., 1998; Weiner etc., 1998).Purifying only comprises TatABC (Bolhuis etc., 2001) from the Tat of intestinal bacteria complex body.Reconstruction in vitro translocation complex proof is to the minimum essential requirement of TatABC and complete film potential (Yahr and Wichner, 2001).

Select leading peptide and thereby can determine whether to produce correct folding functional protein (Bowden and Geogiou, 1990 in the specific egg of output used approach in matter; Thomas etc., 2001).Feilmeier etc. (2000) show that green fluorescent protein (GFP) is fused to the special leading peptide of Sec-or maltose binding protein C-end (MBP is also through the output of Sec approach) causes green fluorescent protein and MBP-GFP to output to pericentral siphon.Yet the green fluorescent protein in the pericentral siphon is non-fluorescence, illustrates that therefore the secretory protein malfolding also can not form the chromophoric group of green fluorescent protein.Owing to folded form transmembrane not, infer that the environment of bacterium secretion compartment (periplasmic space) is unfavorable for that green fluorescent protein folds (Feilmeier etc., 2000) through the protein of Sec approach output.On the contrary, the special leading peptide fusion of Tat-green fluorescent protein causes green fluorescent protein to accumulate in the periplasmic space.In this case, the Tat-GFP propetide at first can fold in tenuigenin, outputs to (Santini etc., 2001 in the periplasmic space as folding fully albumen subsequently; Thomas etc., 2001).Yet, do not have evidence to show that the leading Toplink except TorA is used to make heterologous protein to output to colibacillary periplasmic space.

The cellular compartment that protein folding takes place can have huge effect to biological activity protein output.Bacterial cytoplasm comprises a large amount of protein folding cofactors, and as mate molecule, it promotes folding function and the effect of new synthetic polypeptide controlled by the ATP hydrolysis.On the contrary, bacterial cytoplasm comprises few relatively mate molecule and does not have evidence to show that ATP is present in this compartment.Therefore, numerous protein can not fold and only can reach their native state in the tenuigenin environment in tenuigenin.The unique known method that can secrete folded protein from tenuigenin is by being fused to the special leading peptide of Tat-.Yet, significantly be lower than more widely used Sec approach through the protein outflow of Tat output system.As a result, protein accumulation and the stable state output through the output of Tat approach is lower.

Therefore prior art lacks the method that instructs folded protein effectively to export from tenuigenin.The present invention realizes long-standing needs in this area and ideal.

Summary of the invention

The invention provides the method for separation sequence, sequence can be as the output of leading peptide guidance of heterologous protein matter.The separable leading peptide that can instruct protein of one side of invention to the Tat Secretory Pathway.In addition, the present invention discloses the method for identifying the leading peptide mutant, and the sudden change physical efficiency is improved protein output.

On the one hand, therefore invention provides the method for identifying leading peptide, and leading peptide instructs the protein secreting that improves in bacterium.Therefore in one embodiment, method shown in this paper comprises the sequence that screening can be exported rapidly from the mutant leader peptides library, can protect in the tenuigenin short-lived reporter protein not degraded.Adjusting is through intestinal bacteria double arginine transposition (Tat) approach excretory leading peptide and instruct other Secretory Pathway to separate by method shown in this paper as the leading Toplink of sec approach in the bacterium.Also disclosed the mutant leader peptides sequence of improving output.Mutant leader peptides shows provides significantly higher output of steady-state level, not only is used for short-lived reporter protein, also is used for other stable long-acting protein.

In one aspect of the invention, provide the method for identifying leading peptide, leading peptide instructs the protein output through approach to increase, by way of including but not limited to double arginine transposition (Tat) approach and sec approach.This method can comprise the construction expression box, mutant leader peptides is placed the upstream region of gene of the short-lived reporter protein of coding.Produce the gene that short-lived reporter protein can invest the coding reporter protein by sequence that kytoplasm is degraded.In bacterium, express gained expression cassette, the expression of measuring reporter protein in these bacteriums subsequently.Show and express mutant leader peptides in the cell that reporter protein express to increase and comprise and instruct the leading peptide that protein output increases in the bacterium.The representative leading peptide that top method is identified comprises SEQ ID NO:120-136.

In another aspect of this invention, provide the method that increases polypeptide output through approach, by way of including but not limited to Tat approach and sec approach.This method comprises expression cassette, the mutant leader peptides identified of method shown in this paper is placed the upstream region of gene of coding heterologous polypeptide interested.

In still another aspect of the invention, provide the method screening to suppress or improve the compound of albumen output, by way of including but not limited to Tat approach and sec approach through approach.This method can at first comprise the construction expression box, the mutant leader peptides identified of method shown in this paper is placed the upstream region of gene of the short-lived reporter protein of coding.Produce the gene that short-lived reporter protein can invest the coding reporter protein by sequence that kytoplasm is degraded.In bacterium, express gained expression cassette, the expression of when candidate compound exists or lack, measuring reporter protein in these bacteriums subsequently.The reporter protein that records when candidate compound exists is expressed and is increased the explanation candidate compound and improve protein output, and the reporter protein expression decreased explanation candidate compound arrestin matter output that candidate compound records when existing.

In other one side of the present invention, be provided at the method that produces solvable and biological-active heterologous polypeptide in the bacterium, polypeptide contains a plurality of disulfide linkage.This method can comprise the construction expression box, the leading peptide that instructs protein through the output of double arginine transposition approach is placed the upstream region of gene of coding heterologous polypeptide.Heterologous polypeptide is expressed in the cytoplasmic bacterium of oxidation is arranged subsequently.

Of the present invention other and further aspect, feature and advantage from the description of the present preferable embodiment of following invention obviously.These embodiments are used for disclosing.

The accompanying drawing summary

The feature of foregoing invention, advantage and target and other can be clearer and can understood in detail, above more specific description and some invention embodiments of brief overview set forth in the accompanying drawings.These accompanying drawings form the part specification sheets.Some embodiments of annexed drawings set forth invention and do not think the scope that limits them.

Fig. 1 shows the expression of green fluorescent protein in the different plasmid constructions.Figure 1A shows green fluorescent protein fluorescence minimum in the cell of expressing pGFPSsrA, illustrates that the green fluorescent protein of tenuigenin SsrA-mark almost completely is degraded.Figure 1B shows that green fluorescent protein fluorescence improves in the cell of expressing pTorAGFPSsrA, and the green fluorescent protein output that indication TorA leading peptide instructs improves.Fig. 1 C shows the green fluorescent protein fluorescence in the cell of expressing pTorAGFP.Green fluorescent protein is all expressed in tenuigenin and pericentral siphon.

Fig. 2 shows 6 green fluorescent protein fluorescence among the different clones, and the clone is owing to mutation T orA leading peptide shows the output increase that Tat relies on.

Fig. 3 shows that the pericentral siphon green fluorescent protein among B6 and the E2 clone gathers.Fig. 3 A demonstration is expressed wild-type and is made up (swimming lane 1 and 4), B6 clone (swimming lane 2 and 5) and E2 clone's (swimming lane 3 and 6) the cell pericentral siphon (swimming lane 1-3) and the western blotting of the middle green fluorescent protein of tenuigenin (swimming lane 4-6).GroEL is a tenuigenin mark and DsbA is the pericentral siphon mark.Fig. 3 B shows that the pericentral siphon and the tenuigenin of expressing green fluorescent protein in the cell that wild-type makes up, B6 clones and E2 clones distribute.

Fig. 4 shows that green fluorescent protein fluorescence increases in the cell of expressing wild-type structure, B6, B7 or E2 structure, makes up and merges the green fluorescent protein unmarked, that protein cleavage is stable.

Fig. 5 shows the western blotting of green fluorescent protein in cell pericentral siphon (swimming lane 1-2), tenuigenin (swimming lane 3-4) and the whole cell lysates (swimming lane 5-6) of expressing wild-type structure ( swimming lane 1,3 and 5) or B6 clone ( swimming lane 2,4 and 6).GroEL is a tenuigenin mark and DsbA is the pericentral siphon mark.

Fig. 6 shows the synoptic diagram of the heterodimer output that disulfide linkage links to each other, and wherein only a polypeptide chain merges leading peptide.

Fig. 7 shows pericentral siphon and tenuigenin western blot analysis and AP activity measurement partly in 6 kinds of genetic backgrounds.

Detailed Description Of The Invention

The invention provides the method for identifying and using leader peptide, leader peptide instructs protein secretion raising in the bacterium. Many Plant the protein of tool commercial interest and from bacterium, secrete generation. Yet what numerous protein can not be through bacterium is main Secretory pathway sec approach is effectively exported, and comprises the enzyme in many antibody fragments and some eucaryons source.

Another kind approach of transferring protein from bacterial cytoplasm is called " TAT " (double arginine transposition) approach. Protein is guiding sec mechanism or the TAT approach only depends on leader peptide character, and leader peptide is to be positioned at polypeptide chain The amino acid of general 15-30 initial residue extends. Leader peptide is made up of three zoness of different: (1) amino end End n-zone, (2) hydrophobic core or h-zone and (3) c-stub area.

Plant is to have different and conservative (S/T)-R-R-x-F-L-K (SEQ with the special leader peptide characteristics of protokaryon TAT-ID NO:1) sequence motifs. This sequence motifs is arranged in n-zone/h-known and prediction TAT substrate leader peptide Border (Berks, 1996). Arbitrary arginine residues in the jump signal peptide significantly reduces Protein transfer efficient (Critobal etc., 1999).

The Sec approach is output pathway in the bacterium of the most generally using up to now, relatively be specific to the Sec approach before Lead peptide, the special leader peptide of TAT-is longer 14 amino acid on average, and this is because in n-region extension and the c-zone More polybase residue (Critobal etc., 1999) is arranged. Yet h-zone hydrophobic in the special leader peptide of TAT-is aobvious Work is shorter, and is more because glycine and threonine residues occur.

Wheat pre-23K and pre-Hcf136 two-arginine (RR) motif is for by thylakoid TAT approach target To being necessary; This motif may be the key character of TAT signal. Two-arginine motif is not the TAT-specific target As if to the unique important decisive factor of signal, the other hydrophobic residue behind this motif 2 or 3 residues is also very heavy Want.

Bacterium is two-and but arginine-signal peptide is similar to the target that thylakoid TAT signal and high efficiency instruct TAT to rely on In the plant utricule. Yet, except the hint specific function two-the arginine motif, most bacterium signals Peptide comprises the conserved sequence element. Serious deflection is two-the arginine motif after the phenylalanine of second position, many letters The lysine that number comprises the 4th position. Do not have known thylakoid two-arginine-signal comprises the benzene of this position It is residual that alanine, only one (Arabidopsis P29) comprise after as two-arginine motif the 4th of lysine Base. The definite effect of these high conservative features is unclear, and phenylalanine residue can not had inappropriate effect by leucine Really replace but do not replaced by alanine, illustrate hydrophobicity rather than phenylalanine side chain may be important decision because of Plain. Similarly, replace lysine residue and do not hinder output (Robinson and Bolhuis, 2001).

The protein of exporting through the TAT system at first is folded into their native conformation in cytoplasm, stride subsequently cell Plasma membrane output. Owing to some reasons, the ability of the protein that output has folded in cytoplasm is for commodity production The protein height needs. At first, very rapid folding protein can not pass through more general sec behind the end of synthesis The output pathway secretion. The second, bacterial cytoplasm comprises fully replenishing of folding confactor, and the factor can be assisted newly Give birth to polypeptide and reach its native conformation. On the contrary, the secretion district of bacterium is the folding confactor that comprises seldom, such as the companion Companion's molecule and folding enzymes. Therefore, preferably at first in cytoplasm, take place for generating numerous protein, folding, connect Through the TAT system and output to periplasmic space. The 3rd, co-factor obtains to follow to be folded in the cytoplasm and takes place. As a result, the protein that contains co-factor must be secreted through the TAT approach.

Using protein secreting, particularly TAT output pathway to be used for the restriction of commercial protein production is that the protein mass that can export in this way is less.In other words, the gross protein outflow through the TAT system significantly is lower than the sec approach.

Current, there is not robust techniques to can be used for screening the recombinant protein that Periplasmic secretion increases, there is not the special leading peptide of best TAT-yet.Yet the result that method shown in this paper obtains can determine the feature of best leading peptide, and best leading Toplink is avoided common wild-type or the natural pair-observed low transport speed of arginine leading peptide.The present invention also can be fully and system determine suitably and effectively to export required minimum preamble sequence through the TAT approach.

Therefore, one aspect of the present invention provides the method for identifying leading peptide, leading peptide instructs the protein output through two-arginine transposition or TAT approach to increase, and by the construction expression box, the special leading peptide of sudden change candidate TAT-is placed the upstream region of gene of the short-lived reporter protein of coding.Relatively from the reporter protein molecule of tenuigenin output, this short-lived reporter protein shows the transformation period and reduces in tenuigenin.Can produce short-lived reporter protein, for example invest the gene of coding reporter protein by sequence that kytoplasm is degraded.Generally, mutant leader peptides can be passed through random mutation, fallibility PCR and/or rite-directed mutagenesis generation.In bacterium, express gained expression cassette, the expression of measuring reporter protein in these bacteriums subsequently.Showing the special leading peptide of mutation T AT-of expressing in the cell of reporter protein expression increase can instruct the protein output through the TAT approach to increase.

Method well known to those skilled in the art can be used for making up and contains expression cassette or the carrier of suitably transcribing and translate control signal.Referring to for example, Sambrook etc., 200, " molecular cloning: laboratory manual (the 2nd edition) " (MolecularCloning:A Laboratory Manual (2nd Ed.), Cold Spring Harbor Press, described technology of N.Y..The carrier of invention includes but not limited to plasmid vector and virus vector.

In an embodiment of screening method described herein, green fluorescent protein (GFP) can be used as reporter protein.This method is utilized the special leading peptide secretion of the only available TAT-of functional green fluorescent protein.Yet green fluorescent protein is low and cause the precursor protein (green fluorescent protein that the special leader of TAT-is arranged) of a great deal of to accumulate in the tenuigenin through the special leading peptide output efficiency of TAT-.Tenuigenin green fluorescent protein precursor protein correctly folds and is fluorescence.As a result, cell shows high fluorescence, and part is owing to the tenuigenin precursor and partly owing to secretion, ripe green fluorescent protein in the pericentral siphon cause.Total height fluorescence of these cells causes high background signal, makes separation produce the high effusive leading peptide sudden change complexity of output green fluorescent protein.

For overcoming this problem, can use the green fluorescence reporter protein of fugitive form.This fugitive form is degraded rapidly in bacterial cytoplasm.For example, SsrA sequence A ANDENYALAA (SEQ ID NO:119) but merge green fluorescent protein C-end targeting proteins matter with by ClpXAP protease system degraded (Karzai etc., 2000).As a result, the transformation period of green fluorescent protein was reduced to less than 10 minutes in the tenuigenin from several hours, caused intact cell fluorescence significantly to reduce.

Show when fugitive green fluorescent protein merges the special leading peptide of wild-type TAT-, observe low-level cell fluorescence, because most of protein is being degraded before tenuigenin output.The sudden change of the special leading peptide of expection TAT-can cause faster and more effective output, and fugitive green fluorescent protein is not degraded in the protection tenuigenin.As a result, folding green fluorescent protein can accumulate in the pericentral siphon, produces higher cell fluorescence.Therefore, the library of the special leading peptide of mutation T AT-makes up by random mutation (fallibility PCR) or Nucleotide rite-directed mutagenesis.Screen these mutant leader peptides subsequently and regulate the ability that fugitive green fluorescent protein is not degraded in protein secreting raising and the protection tenuigenin, therefore cause bacterial fluorescence to increase.The clone who shows higher fluorescence then separates by flow cytometer.

A special characteristic of the present invention is that genetic screening described herein makes active reporter protein only accumulate in pericentral siphon.Mutant leader peptides instructs folding green fluorescence reporter protein to keep active pericentral siphon to fluorescin.Yet, owing to have the terminal degraded of SsrA C-peptide, nearly all tenuigenin green fluorescent protein degraded.Because green fluorescent protein exists only in pericentral siphon, the gained cell is luminous in the ring of light-type mode.On the contrary, the green fluorescent protein that TAT relies on output shortage SsrA sequence can cause green fluorescent protein to accumulate in tenuigenin and the pericentral siphon, produces remarkable background signal, and making with the cell is that basis screening 6FP fluorescence is impossible.

Except green fluorescent protein, multiple other reporter protein can be used for method of the present invention.Those of ordinary skills can easily separate mutant leader peptides, and mutant leader peptides produces higher levels of reporter protein with certain methods and expresses in pericentral siphon.In an example,, separate mutant leader peptides and can select by increasing antibiotic concentration if reporter molecules is antibiotics resistance enzyme (as a β-Nei Xiananmei).In another example,, separate mutant leader peptides and can select by the toxin resistance if reporter molecules is the immune protein of toxin (as Colicine).In another example, if reporter protein is translocator such as maltose binding protein, translocator output is used for the sudden change of complementary stain body.In the another one example, the color development of reporter enzyme (as alkaline phosphatase) or fluorescence produce substrate can be used for estimating the bacterium colony that produces the higher level enzyme at the bacterium pericentral siphon.

There are some researchs and industry to be used for screening system described herein.The example that these researchs and industry are used comprises but must not be limited to following:

(1) the biological protein that generates: secreting some protein through the TAT approach is slow relatively and inefficient method by report.Therefore, must realize improving the needs of output so that the TAT approach becomes the feasible platform that high level generates high value recombinant protein product.Use the generalized genetic screening of this paper, separate best TAT leading peptide and test their abilities of output recombinant protein interested rapidly.Therefore, recombinant protein is secreted into periplasmic space or substratum with function or soluble form, alleviates the inclusion body associated problem and simplifies recovery.In addition, because protein is folding and accumulate in tenuigenin before the output that TAT relies on, this output system may cause that higher levels of active result gathers in the host cell, thereby the efficient of maximization recombinant expression system.

(2) in the high flux screening platform: the present invention can be used for development utilization TAT and relies on the technology of output to use in conjunction with library screening and protein engineering.For example, the folding improvement of tenuigenin that contains disulfide bond protein matter (as antibody, eucaryon enzyme) can be measured by being fused to best leading peptide, best leading peptide is exported folded protein interested to pericentral siphon, and it can be by based on the screening of FACS-or phage acquisition simple to operate therein.Detected activated protein amount is the quantitative indication of folding efficiency in the tenuigenin in the pericentral siphon.

(3) in drug discovery programs: the proteic homologue of some TAT is found in malignant bacteria, as mycobacterium tuberculosis (Mycobacterium tuberculosis) and Hp (Helicobacter pylori) and Rhodopseudomonas.Some protein that this explanation belongs to this transposition system can be the potential novel targets of antiseptic-germicide.Use the generalized method of this paper, can easily screen a large amount of compounds to suppress the secretion of TAT dependent form.In addition, obtaining in the multiple copied of genomic library, to exist some protein or missing gene can be with new enhanser/inhibitions of this method test to identify TAT secretion process in the bacterium from genome at random, thereby the more universal method that develops antiseptic-germicide is being provided.

Identify and use the TAT approach that the invention is not restricted to of leading peptide, leading peptide instructs protein secreting raising in the bacterium.Method shown in this paper can be applicable to identify the leading peptide that instructs protein secreting to improve through above-mentioned other Secretory Pathway equally.The signal sequence that promotes protein to translocate to the Gram-negative bacteria periplasmic space is known those skilled in the art.For example, colon bacillus OmpA, Lpp, Lamb, MalE, PelB, StII leader peptide sequences successfully are used for many application as the signal sequence that promotes the bacterial cell protein secreting, as used herein, expect that they can both be used to put into practice the inventive method.Those of ordinary skills can easily use well known process with structure sudden change leader sequence library and the expression cassette that comprises these mutant leader peptides, and screen these leading peptides according to methods described herein.

The present invention also relates to the proteic secretion of partially or completely folded cell matter of tool disulfide linkage.The formation disulfide linkage is essential to the correct folding and stability of multiple eukaryotic protein, and these protein are important to medicine and biological processing industry.Correct folding the depend on formation of halfcystine-halfcystine key and the protein stability that enters the enzymic activity structure subsequently.Yet, manyly studies have shown that the multiple protein that contains disulfide linkage can not express with activity form in bacterium.Because existing thioredoxin reductase or reduced glutathion, disulfide linkage to be formed in the reducing environment of bacterial cytoplasm is hindered.

Therefore, generation has the technical key protein cost of four or more disulfide linkage big and complicated, must depend on to express in the higher eucaryote that provides disulfide linkage to form enabling environment or from inclusion body folding again (Hockney, 1994, Georgiou and Valax, 1996).For example, tissue plasminogen activator (tPA) generates in the bacterium inclusion body at present.In typical process, protein discharges from inclusion body with multiple chaotropic agent, and is separated subsequently and folding again with reductive agent.Generally, foldingly again cause biologically active substance to yield poorly.

Secretion method described herein provides the method for the complicated eukaryotic protein of a plurality of disulfide linkage of effective generation tool.These disulfide linkage form to promote the correct folding of natural protein from specific direction.A plurality of disulfide linkage that the inappropriate direction of nascent protein produces in the cell cause malfolding and lose or lack biological activity.On the contrary, a plurality of disulfide linkage that contain biologically active polypeptides that generate according to instant invention correctly fold; Form disulfide linkage tertiary structure to be provided and to provide quaternary structure in applicable place, the molecule of generation has natural function activity and/or the catalytic property about substrate.The protein that produces by methods described herein is correctly folding and biological activity is arranged, and does not need reactivate or processing subsequently in case separate from host cell.

The immediate problem that methods described herein solve be a plurality of disulfide linkage of tool protein now can with fully folding and thereby active conformation output to pericentral siphon.The complex proteins that contains a plurality of disulfide linkage can be folded in the tenuigenin under the assistance that fully replenishes folding cofactor, and cofactor promotes newborn polypeptide to reach their native conformation.Folded protein is secreted into periplasmic space or substratum with function and soluble form subsequently, therefore alleviates the inclusion body associated problem and simplifies recovery.In addition, active recombinant protein accumulates in two bacterium compartments (tenuigenin and pericentral siphon) simultaneously, makes the overall productivity of multiple complex proteins higher, can not while activity accumulation in two compartments before the complex proteins.

Therefore, the invention provides the method that in cell, produces at least a biological activity heterologous polypeptide.Instruct protein can place the upstream region of gene of expression cassette coding heterologous polypeptide through the leading peptide of double arginine pathway output.Expression cassette can be expressed in cell, and wherein heterologous polypeptide produces with biologically active form.Generally, heterologous polypeptide is secreted from bacterial cell, can from the pericentral siphon of bacterial cell or culture supernatant, separate, or conformity membrane albumen.The heterologous polypeptide that this method produces can be the Mammals polypeptide, as tissue plasminogen activator, pancreatic trypsin inhibitor, antibody, antibody fragment or toxin immunity albumen.Heterologous polypeptide can be polypeptide, mutant polypeptide or the brachymemma polypeptide of native conformation.

Make the cytoplasmic cell of apparatus oxidation, the heterologous polypeptide that aforesaid method can produce contains about 2 to about 17 disulfide linkage.This method also can produce two kinds of heterologous polypeptides that linked to each other by at least one disulfide linkage.Leading peptide preferably include SEQID NO:25-46,120-128 sequence or with SEQ ID NO:25-46,120-128 homologous peptide.The representative cell that is used for this method comprises intestinal bacteria trxB mutant strain, intestinal bacteria gor mutant strain or intestinal bacteria trxBgor double-mutant strain such as coli strain FA113 or coli strain DR473.

The present invention also provides a series of TAT-of inferring special leading peptide, can identify by searching for from the intestinal bacteria bioinformation, clone and the measuring ability activity.Therefore, the leading peptide that the present invention includes instructs protein through double arginine pathway secretion and output.Represent leading peptide to comprise the sequence of SEQ ID NO:25-46,120-128.In addition, the present invention includes with SEQ ID NO:25-46,120-128 homologous and separate the TAT leading peptide.

The present invention also provides method to identify and instructs the leading peptide that protein is exported to be increased, mutant leader peptides is placed the upstream region of gene of the short-lived reporter protein of coding by the construction expression box.Produce the gene that short-lived reporter protein can invest the coding reporter protein by sequence that kytoplasm is degraded.Represent kytoplasm degraded sequence to comprise SEQ ID NO:119, PEST or the sequence of discerning by LON, clPAP, clPXP, Stsh and HslUV.Kytoplasm degraded sequence invests the N-or the C-end of reporter protein.Generally, spendable reporter protein comprises fluorescin, enzyme, translocator, antibiotics resistance enzyme, toxin immunity albumen, phage receptor protein and antibody.

Can produce and use mutant leader peptides, for example by random mutation, fallibility PCR or rite-directed mutagenesis and method known to those skilled in the art.Can in bacterium, express gained expression cassette, the proteic expression of measurement report subsequently.Show the special leading peptide of mutation T AT-of expressing in the cell of reporter protein expression increase and comprise the leading peptide that can instruct protein output increase in the bacterium.The leading peptide that this screening method can be identified instructs protein to rely on approach or the secretion of double arginine transposition (Tat) approach through general secretion (Sec) approach, signal recognition particle (SRP) dependence approach, YidC.

In another aspect of this invention, provide the method that increases heterologous polypeptide output in the bacterium.The construction expression box will place the encoding sequence upstream of coding heterologous polypeptide interested according to the inventive method mutant leader peptides identified.These expression cassettes can be expressed in bacterium subsequently.

The present invention also provides the method screening to suppress or improve the compound of protein output in the bacterium.Instruct the leading peptide of the output of protein in the bacterium to place the upstream region of gene of the short-lived reporter protein of expression cassette coding.Expression cassette can be expressed in the bacterium when test compounds exists or lack subsequently.The reporter protein that records when test compounds exists is expressed and is increased the explanation compound and improve protein output, and the reporter protein expression decreased explanation compound arrestin matter output that test compounds records when existing.The structure and the example of short-lived reporter protein are described in.

The leading peptide that the present invention also provides method to identify to instruct protein to increase through the output of double arginine transposition approach, the mutant leader peptides that will be specific to double arginine transposition approach by the construction expression box places the encoding sequence upstream of the short-lived reporter protein of coding.The structure and the example of short-lived reporter protein are described in.Mutant leader peptides can be passed through random mutation, fallibility PCR or rite-directed mutagenesis and produce.The gained expression cassette can be expressed in bacterium subsequently, the proteic expression of measurement report.Show the special leading peptide of mutation T AT-of expressing in the cell of reporter protein expression increase and comprise the leading peptide that can instruct protein to increase through the output of double arginine transposition approach.The example of mutant leader peptides comprises the sequence of SEQ IDNO:120-128.

In still another aspect of the invention, providing method to increase heterologous polypeptide exports through double arginine transposition approach.The construction expression box will place the upstream region of gene of coding heterologous polypeptide interested according to method mutant leader peptides identified shown in the invention.These expression cassettes can be expressed in bacterium subsequently.The example of mutant leader peptides comprises the sequence of SEQ ID NO:120-128.

The present invention also provides the method screening to suppress or improves the compound of protein through the output of double arginine transposition approach.The leading peptide that is specific to double arginine transposition approach can place the upstream region of gene of the short-lived reporter protein of expression cassette coding.Expression cassette can be expressed in the bacterium when test compounds exists or lack subsequently.The reporter protein that records when test compounds exists is expressed and is increased the explanation compound and improve protein output, and the reporter protein expression decreased explanation compound arrestin matter that compound records when existing is exported through double arginine transposition approach.The structure and the example of short-lived reporter protein are described in.

As used herein, " polypeptide " or " polypeptide of interest " refers generally to have above 10 amino acid whose peptides and protein.Polypeptide is " allogenic ", refers to that they are external sources to used host cell, the yeast polypeptides that human protein that produces as Chinese hamster ovary celI or Mammals produce, the human polypeptides that is produced by the human cell line in non-natural polypeptide source.The example of polypeptide of interest includes but not limited to molecule such as feritin, tethelin (comprising human growth hormone), Trobest, somatotropin releasing factor, Rat parathyroid hormone 1-34, thyrotropic hormone, lipoprotein, alpha1-antitrypsin, INSULIN A-chain, the Regular Insulin beta chain, proinsulin, thrombopoietin, follicle stimulating hormone, calcitonin, lutropin, hyperglycemic-glycogenolytic factor, thrombin is (as Factor IX C, IX, the tissue factor and the vonWillebrands factor), anticoagulin is (as protein C, the heart is received element, Curosurf), plasminogen activator (as buman tPA or urokinase), the Mammals trypsin inhibitor, brain derived neurotrophic growth factor, kallikrein, CTNF, gp120, anti-HER-2, human chorionic gonadotropin, the mammalian pancreas trypsin inhibitor, antibody, antibody fragment, proteinase inhibitor, the treatment enzyme, lymphokine, cytokine, somatomedin, neurotrophic factor, Regular Insulin chain or proinsulin, immunotoxin, Magainin, zymoplasm, tumor necrosis factor-alpha or β, enkephalinase, serum albumin (as human serum albumin), Miller pipe inhibition, relaxin A-chain, relaxin B-chain, relaxin is former, mouse gonad-stimulating hormone related peptides, microprotein (as β-Nei Xiananmei), the DNA enzyme, statin, activin, blood vessel endothelial factor (VEGF), hormone or growth factor receptors, integral protein, a-protein or D, Rheumatoid factors, polyclonal, neurotrophic factor is (as neurotrophin-3,-4,-5 or-6), or nerve growth factor (as NGF-β), heart nutrient protein (the cardiac hypertrophy factor) (as heart nutrient protein-1), Thr6 PDGF BB (PDGF), fibroblast growth factor (as α FGF and β FGF), Urogastron (EGF), transforming growth factor (TGF) is (as TGF-α and TGF-β 1, TGF-β 2, TGF-β 3, TGF-β 4 or TGF-β 5), insulin-like growth factor I and II, des (1-3)-IGF-I (brain IGF-1), insulin-like growth factor binding protein, CD albumen is (as CD-3, CD-4, CD-8 and CD-19), erythropoietin, bone-inducing factor (osteoinductive factor), bone morphogenetic protein (BMP), Interferon, rabbit is (as interferon-' alpha ',-β and-γ), G CFS (CSF) is (as M-CSF, GM-CSF and G-CSF), interleukin (Ils) (arriving IL-10) as IL-1, superoxide dismutase, T-cell receptors, surface membrane protein, decay accelerating factor, virus antigen such as AIDS coating part, translocator, homing receptor, addressin, regulate albumen, antigen such as gp120 (IIIb), or the derivative of any above-mentioned peptide or active fragments.Polypeptide can be natural or mutant polypeptide, and the preferred source of this Mammals polypeptide comprises people, ox, horse, pig, wolf and rodent source, and people's albumen is especially preferred.

Following example is used to illustrate the multiple embodiments of invention and does not think to limit by any way the present invention.

Embodiment 1

The bioinformation search of the special leading peptide of TAT-

Infer the TAT leading peptide and find that with protein-protein " BLAST " search engine search engine can obtain the network address from NCBI.Key in following search string: SRRRFLK (SEQ ID NO:2), SRRXFLX (SEQ ID NO:3), TRRXFLX (SEQ ID NO:4), SRRXXLK (SEQ ID NO:5), SRRXXLA (SEQ ID NO:6), TRRXXLK (SEQ ID NO:7), TRRXXLA (SEQ ID NO:8), SRRXXLT (SEQ ID NO:9), SRRXXIK (SEQ ID NO:10), SRRXXIA (SEQ ID NO:11), SRRXFIX (SEQ IDNO:12), SRRXFMK (SEQ IDNO:13), SRRXFVK (SEQ IDNO:14), SRRXFVA (SEQ ID NO:15), SRRQFLK (SEQ ID NO:16), RRXFLA (SEQ ID NO:17) and RRXFLK (SEQ ID NO:18).Weak point, approximate accurate pairing are searched for, only screen subsequently and in preceding 50 residues of protein, take place and still keep the pairing of double arginine.Use " SignalP " to detect preceding 100 residues of each leading peptide then, this is the program (Nielsen etc., 1997) that detects Sec approach leading peptide and cleavage site.The ultimate sequence of inferring the TAT leading peptide is shown in table 1.Clone these peptides and detect them and instruct reporter protein GFP-SsrA through TAT approach excretory ability.

Bacterial isolates and growth conditions:

Cell is having on suitable antibiotic solid LB agar or the liquid LB substratum always 37 ℃ of growths.Use the paraxin (Cm) of 50 μ g/mL concentration.Strain X l1-Blue (recA1 endA1 gyrA96 thi-1 hsdR17supE44 relA1 Lac[F ' roAB lacl ^qZ Δ M15Tn10 (TeT ^г)]) (Stratagene) be used to clone purpose.In order to express, high copy pBAD18-Cm makes up and is transformed into bacterial strain MC4100-P (MC4100pcnB1) and B1LK0-P (MC4100 Δ tatCpcnB1).

Plasmid and oligonucleotide:

Respectively infer at first subclone (Delisa etc., 2002) in pKKGS of leading peptide dna sequence dna, pKKGS is based on low copy pBAD-33 plasmid (Guzman etc., 1995).Use standard method DNA amplification and Qiagen test kit to be used to all DNA purification steps.Each leading peptide gene is pcr amplification from the Xl1-Blue genomic dna at first, with forward primer that contains the SacI cleavage site and the reverse primer that contains the XbaI cleavage site.The design forward primer comprises preceding 18 Nucleotide of leading peptide at least.All forward primers comprise sequence (5 '-GCGATGGAGCTCTTAAAGAGGAGAAAGGTC-3 ', SEQ ID NO:19), then are initiator codons and from the leader peptide sequences of required gene.Similarly, all reverse primers comprise sequence (5 '-GCGATGTCTAGA-3 ', SEQ ID NO:20).The design reverse primer makes 6 definite amino-acid residues behind the prediction leading peptide cleavage site include plasmid in.58 primers of gained are shown in table 2 and 3.All PCR products digest with glue purification and with SacI and XbaI, are cloned into SacI and the XbaI site of Pkkgs at last.All plasmid constructions are confirmed by order-checking.

Similar structure produces (Guzman etc., 1995) with high copy pBAD18-Cm.Briefly, signal sequence-GFP-SsrA constructs the same loci that digests and be cloned into pBAD18-Cm with SacI and HindIII from pBAD33.In the situation of HybO leading peptide, pBAD18-Cm is cut and be cloned into to the HybO-GFP-SsrA fusion with SacI and SphI.All plasmid constructions such as preceding by the order-checking affirmation.

Proteinic Subcellular Localization:

Cell is resuspended to 1ml cell grade damping fluid (30mlTris-HCl, pH8.0,20% (w/v) sucrose, 1mM Na by 5000x g centrifugation ₂EDTA), hatched 10 minutes for 25 ℃.Cell is centrifugal with 5000x g once more, abandons supernatant, and pellet resuspended is in the ice 5mM of 133 μ l MgSO ₄After 10 minutes, cell is centrifugal at full speed on ice, keeps supernatant as the pericentral siphon part.Pellet resuspended was in 250 μ l PBS and ultrasonication 30 seconds.Cell is centrifugal at full speed and keep supernatant as the tenuigenin part.

Western blot analysis:

Western blotting is according to Chen etc.Use following initial antibody: the mono-clonal mouse anti GFP (Clontech), 1: 10 of dilution in 1: 5000, the anti-DsbC of mono-clonal rabbit (John Joly, Genentech gives) of 000 dilution and 1: 10, the mono-clonal rabbit of 000 dilution resists-GroEL (Sigma).Secondary antibodies is 1: 10,000 goat anti-mouse-HRP conjugate and goat antirabbit-HRP conjugate.Film is at first surveyed with anti-GFP and anti-DsbC antibody, and slough in the TBS/2%/SDS/0.7M beta-mercaptoethanol back of developing.The film of wash-out heavily seals and surveys with anti-GroEL antibody.

Leading peptide is inferred in the FACS screening:

Make up for expressing leading peptide-GFP-SsrA, contain the MC4100-P of each 30 plasmid and (o/n) culture that spends the night of B1LKO-P and grow in as mentioned above in the LB substratum.Single bacterium colony overnight growth in the 2mL substratum.Each o/n culture of 500 μ l is used to inoculate the 10mL substratum.37 ℃ shake 1h after, cell is induced with the pectinose of final concentration 0.02%.37 ℃ cultivate 4 hours again after, collect the 1mL sample and centrifugal 5 minutes with 2500x g.Cell precipitation is resuspended to 1mL PBS.5 μ l wherein add the new PBS of 1mL and are analyzed by Becton-DickensonFACSort.

Infer the screening (DeLisa etc., 2002) on the heredity screen as mentioned above of TAT leading peptide for 30 kinds.By this genetic screening, instruct GFP through the leading peptide of TAT approach at tatC ⁺Fluoresce in the cell (MC4100-P), but at tatC ^-The no fluorescence of cell (B1LKO-P) is because TAT output absolute demand tatC.On the contrary, instruct GFP in two kinds of cells, all not have fluorescence to the leading peptide of pericentral siphon through the Sec approach.Notice that used coli strain contains the pcnB1 sudden change, reduces these plasmid that contains the pBR322 replicon (as pBAD18-Cm) copy numbers.Therefore, normally only be present in the pcnB1 mutant strain with about 5-10 the every cell of copy for the pBAD18-Cm of high copy vector.This system's proof is to using TAT approach genetic screening the best.

The facs analysis that pBAD18-Cm makes up is shown in table 4 (fluorescence arithmetical mean value list).Importantly, 6 kinds of leading peptides of FACS data presentation (BisZ, NapA, NapG, YaeI, YgfA and YggJ) that pBAD18-Cm makes up can not determine GFP through TAT approach output (signal is all low in wild-type and tatC mutant cell), and at least 17 kinds (AmiC, DmsA, FdnG, FdoG, FhuD, HyaA, HybA, NrfC, SufI, TorA, WcaM, YacK, YahJ, YdcG, YdhX, YfhG and YnfE) can export through the TAT approach.5 kinds of structures (YagT, YcbK, YcdB, YedY and YnfF) all show very high fluorescence mean number in MC4100-P and B1LKO-P.Some that also it should be noted that tatC mutant (B1LKO-P) make up that viewed higher mean fluorecence signal only reflects from the high fluorocyte group's of sub-fraction emission and most of cell mass is non-fluorescence.On the contrary, the high mean fluorecence of tatC+ cell (MC4100-P) is indicated the transfer of fluorescent emission in the colony.

Table 1: the special leading peptide of intestinal bacteria TAT-

* the amino acid of emphasizing with grey constitutes the double arginine consensus motif.

Each forward primer of the special leading peptide of table 2:29 kind TAT-and their melting temperature(Tm)

Each reverse primer of the special leading peptide of table 3:29 kind TAT-and their melting temperature(Tm)

Table 4: the FACS screening of inferring leading peptide

The fluorescence arithmetical av is from pBAD18-Cm: in MC4100-P and the B1LKO-P cell: the FACS data that leading peptide-GFP-SsrA makes up.The data computation of B1LKO-P cell is from all cells (showed cell %), except the high fluorocyte group of small portion.

Embodiment 2

Bacterial isolates and plasmid construction

All bacterial strains and the plasmid that use in the following example are listed in table 5.Except as otherwise noted, coli strain XL1-Blue (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 Lac[F ' proAB lacl ^qZ Δ M15Tn10 (Tet ^г)]) be used for all experiments.Intestinal bacteria XL1-BluetatB and XL1-Blue tatC use pFAT24 (Sargent etc. 1999) and pFAT166 (Bogsch etc., 1998) process (Bogsch etc., 1998) generation according to the rules respectively.The bacterial strain routine is longer than in Luria-Bertani (LB) substratum and microbiotic adds with following concentration: ampicillin, 100 μ g ml at 37 ℃ of aerobes ^-1, paraxin, 25 μ g ml ^-1

The plasmid that makes up in the following example produces (Sambrook etc., 2000) based on pBAD33 (Guzman etc., 1995) and with standard operation.Make up plasmid pGFP by clone GFPmut2 variant (Crameri etc., 1996), use primer GFPXbalI (5 '-GCGATGTCTAGAAGTAAAGGAGAAGAACTTTTC ACT-3 ', SEQ ID NO:112) and GFPHindIII (5 '-GCGATGAAGCTTCTATTTGTATAGTTCATCCAT-3 ', SEQ ID NO:113), they import unique restriction site of XbalI and HindIII respectively 5 ' and 3 ' end of 716-bp gfpmut2 gene and this sequence can be cloned in the plasmid DNA of XbalI and HindIII digestion.Plasmid pGFPSsrA produces similar, use primer GFPXbalI and GFPSsrA (5 '-GCGATGAAGCTTGCATGCTTAAGCTGCTAAAGCGTAGTTTTCGTCGTTTGCTGCGT CGACTTTGTATAGTTCATCCATGCC-3 ', SEQ ID NO:114) to import unique SsrA recognition sequence.Plasmid pTorAGFP and pTorAGFPSsrA produce by pcr amplification bacillus coli gene group DNA, use primer TorASacI (5 '-GCGATGGAATTCGAGCTCTTAAAGAGGAGAAAGGTCATGAACAATAACGATCTCTT TCAG-3 ', SEQ ID NO:115) and TorAXbalI (5 '-GCGATGTCTAGAAGCGTCAGTCGCCGCTTGCGCCGC-3 ', SEQ ID NO:116) to produce the torA cDNA of 138-bp, at 5 ' and 3 ' end SacI and XrbalI restriction site are arranged respectively.This sequence is inserted the pGFP or the pGFPSsrA plasmid DNA of SacI-XbalI digestion subsequently.All plasmids that make up in this research are confirmed by order-checking.

Table 5: bacterial isolates and plasmid

Embodiment 3

Flow cytometry analysis

The XL1-Blue cell has the plasmid based on GFP, and its overnight culture goes down to posterity in the fresh LB substratum of cultivating paraxin and induces with 0.2% pectinose when middle exponential phase growth.After 6 hours, cell is washed once with PBS, and the cell dilution that 5 μ l washed is analyzed with Becton-Dickinson FACSort in 1ml PBS then.

Embodiment 4

Produce the torA combinatorial library

Random mutation strain library makes up by the fallibility PCR of torA gene order, use 3.32 or 4.82mMMg ²⁺(Fromant etc., 1995), XL1-Blue genomic dna and following primer: torASacI (5 '-GCGATGGAATTCGAGCTCTTAAAGAGGAGAAAGGTCATGAACAATAACGATCTCTT TCAG-3 ') (SEQ ID NO:117) and torAXbalI (5 '-GCGATGTCTAGAAGCGTCAGTCGCCGCTTGCGCCGC-3 ') (SEQ ID NO:118).For making up error rate is 0.5% library, use 0.22mM dATP, 0.20mM dCTP, 0.34mM dGTP and 2.36mM dTTP, and 0.12mM dATP, 0.1mM dCTP, 0.55mM dGTP and 3.85mM dTTP to be used to make up error rate be 1.5% library.The library places gfpssrA sequence upstream with SacI-XbalI digestion and at the pGFPssrA that is connected between SacI-XbalI with the library.The reaction mixture electroporation is to electroreception attitude XL1-Blue cell (Stratagene), and the serial dilution thing is cultivated on the selectivity flat board to determine the quantity of independent transformant.

Embodiment 5

Library screening

Transformant is 37 ℃ of growths in the LB substratum of paraxin is arranged, with 0.2% pectinose induced 6 hours and in 1ml PBS the dilution 200 times.FACS door (gate) is provided with on FSC/SSC and FL1/FL2 basis.Before the classification, the library cell mass uses the iodate third ingot mark with the non-survivaling cell of preferred mark.Ca.3 * 10 altogether ⁶Individual cell is by flow cytometry analysis and collect 350 survivaling cells.Filter the solution of collecting, filtrate places on the LB flat board of paraxin.Cultivate after 12 hours for 37 ℃, among the LB in colony inoculation to three 96 orifice plate, paraxin is arranged among the LB separately.37 ℃ of growths are after 12 hours, and cell goes down to posterity similarly and cultivates in three times of 96 orifice plate, and plate comprises the LB and 0.2% pectinose of tool paraxin, 37 ℃ of growths 6 hours.Separately the clone through FACS and fluorescence plate reader (Bio-Tek FL600, the Bio-Tek instrument, Winooski, VT) screening is used to confirm the fluorescence phenotype.

Embodiment 6

Cell grade separates

Periplasm protein partly obtains by the bacterium spheroplast, is waiting under the condition of oozing according to the process of Kaback (1971) with N,O-Diacetylmuramidase-EDTA processing.Briefly, cell is by centrifugal collection and be resuspended to OD in damping fluid ₆₀₀Be 10, damping fluid contains 100mM Tris-Cl (pH8.0), 0.5M sucrose and 1mM Na-EDTA.N,O-Diacetylmuramidase (Sigma) adds to 50 μ g/ml, cell incubated at room 1 hour to produce spheroplast.Spheroplast is by 3, and 000x g collected in centrifugal 15 minutes, collects the supernatant that contains periplasm protein and is used for electrophoretic analysis.The pellet resuspended that contains spheroplast in 10ml TE (10mM Tris-Cl[pH7.5], 2.5mM Na-EDTA) and in French press cell (Carver) with 2,000lb/in ²Stir evenly.For analyzing the gross protein of untreated cell, directly be resuspended to 10mlTE, then carry out French pressure homogenizing.

Embodiment 7

The improved phenotype of output in the screening signal peptide library

Plasmid pTorAGFP comprises the gene of the special leading peptide of coding TAT-and preceding 8 amino acid of intestinal bacteria Trimethylamine 99-N-oxide compound reductase enzyme (TorA), the best GFPmut2 gene fusion (Crameri etc., 1996) of amino acid and FACS.The TorA-GFP gene places pectinose inducible promoter pBAD downstream.Cell is induced with pectinose and by facs analysis, the average fluorescent strength of generation (MFL1) is greater than 500 arbitrary units (Fig. 1 C).Consistent with the report of front (Santini etc., 2001), the cell grade by osmotic shock separate that the total fluorescence that shows ca.40-50% is positioned at the wild-type cell pericentral siphon and tenuigenin GFP accounts for the total fluorescence of remaining 50-60%.In the tatB and tatC mutant strain that remove the TAT approach, the total fluorescence above 95% remains in the tenuigenin, thereby proof TorA-GFP exports through the TAT approach.

The nucleotide sequence and the TorA-GFP gene fusion of the terminal SsrA degraded of coding C-peptide.Gained gene pTorA-GFP-SsrA also places the pBAD promotor downstream of carrier pTorAGFPSsrA.As negative contrast, do not have the GFP and the SsrA indicia framing endomixis of leading peptide and from plasmid pGFPSsrA, express.The cell of expressing GFP-SsrA does not almost show tangible fluorescence intensity, the GFP that tenuigenin SsrA-mark is described almost completely degrade (Figure 1A).Express the cell of TorA-GFP-SsrA and compare the cell of expressing GFP-SsrA, the high ca.8 of fluorescence is (Figure 1B) doubly.In tatB and tatC mutant cell, express TorA-GFP-SsrA and only cause background fluorescence.

Fallibility PCR (Fromant etc., 1995) is used to produce TorA leading peptide random mutation library.The mutation frequency that estimate in 3 libraries that make up is that 0.5,1.5 or 3.5% Nucleotide replaces.Mutation T orA leading peptide is connected into the GFP-SsrA upstream of pGFPSrA.The library that transformed into escherichia coli produces is by 10 ⁶To 10 ⁷Individual independent transformant is formed.20 clones that select immediately of sequential analysis confirm to exist in the TorA leading peptide sudden change that distributes immediately.

With FACS is that 6 clones are altogether isolated in 3 libraries of screening, basis, 2 library and 4 libraries from low error rate from higher error rate.All 6 the relative parental generation TorA-Gfp-SsrA of clone make up and show higher cell fluorescence (Fig. 2).Relative wild-type leading peptide, fluorescence level increases by 3 to 6 times.Oppositely transform these and be cloned into strain X L1-Blue or DHB4 causes fluorescence level stable, give rather than because incoherent sudden change in the host cell thereby explanation fluorescence increases by each plasmid.When plasmid was transformed into tatB and tatC cell, cell fluorescence disappeared, as depended on that the process of TAT output system expects.

Pericentral siphon GFP gathers the remarkable increase (Fig. 3, swimming lane 1-3) that relative expression's wild-type makes up in typical case's western blotting explanation expression B6 and E2 clone's the cell.In addition, almost there is not detectable GFP albumen in the tenuigenin part.This is because the existence of SsrA mark causes protein degradation.Fig. 3 also shows the Western band of two portions labelled protein, i.e. tenuigenin mark GroEL and pericentral siphon mark DsbA.Pericentral siphon partly lacks GroEL and has high-level DsbA proof cell grade to separate successfully.

The fluorescence partial data is shown in Fig. 3 B in the tenuigenin of two kinds of mutation T orA leading peptides and the pericentral siphon part.Remain the result who observes among 4 clones (B7, F1, F11 and H2) much at one.Determine 6 clones' sequence, illustrate that 1 or 2 single residue sudden change is enough to change observed outputting power in all situations.Generally, these sudden changes occur in conservative S/T-R-R-x-F-L-K (the SEQ ID NO:1) consensus motif or very near (table 6).

Confirm that 6 kinds of mutation T orA leading peptides increase GFP output, not only at protein during with SsrA sign mark but also to the GFP unmarked, that protein cleavage is stable also so (Fig. 4).This fluorescence increase is because the proteic pericentral siphon of folding GFP flows out increase.Observe similar results for the residue clone of merging GFP.

The tenuigenin GFP level of the cells accumulation of two kinds of structures of typical western blotting explanation expression of comparison wild-type TorA-GfP and TorAB7-Gfp is (Fig. 5, swimming lane 3 and 4) much at one.Yet the amount of output GFP is at the cell significantly higher (Fig. 5, swimming lane 1 and 2) of expressing the TorAB7-GFP clone.Its further evidence can be found in the intact cell lysate.The strong band that is expressed as maturation (M) GFP is represented the TorA-Gfp chimeric protein, and this albumen is most possibly by signal peptidase I processing (Berks etc., 2000).Therefore, the strong band that makes up the ripe GFP gather corresponding to TorAB7-Gfp illustrates that the GFP of its pericentral siphon processing is significantly greater than wild-type TorAGFP cell (Fig. 5, swimming lane 5 and 6).Observe similar results for all 5 remaining clones.As mentioned above, GroEL confirms that with the DsbA labelled protein successful cell grade separates.

6:6 in table shows the cloned sequence that the secretion of TAT dependent form increases

The double arginine consensus motif is represented by underlined amino acid; Proteic preceding 8 residues of mature T orA are represented with italic; The sudden change of TorA leading peptide is represented with boldface letter.

Embodiment 8

The folding recombinant protein that contains a plurality of disulfide linkage is secreted through double arginine transposition output pathway

An embodiment of method shown in this paper comprises the fusion of using between special leading peptide of TAT-and heterologous polypeptide interested.For example, the special leading peptide TorA of TAT-can merge alkaline phosphatase (TorA-PhoA fusion).Alkaline phosphatase (PhoA) comprises two disulfide linkage, disulfide linkage in primary sequence continuously thereby they can not in coli strain (as the bacterial strain DHB4) tenuigenin of tool reducing environment, form usually.Because the TAT approach need fold or partially folded at least substrate, depend in the DHB4 cell that the PhoA secretion of TAT is hindered owing to fold gathering of PhoA in the tenuigenin.

Therefore, tenuigenin need be become the state of oxidation to secrete through the TAT approach.Usually, bacterial cytoplasm is kept reduced state, because have reduction composition such as gsh and Trx, is unfavorable for forming in the protein very much disulfide linkage.Bacterial isolates is transformed in the early stage work of Bessette etc., and bacterial strain has highly oxidized tenuigenin, can effectively form disulfide linkage (Bessette etc., 1999).Shown in Bessette etc., the aerobic growth when intestinal bacteria rely on one or both main mercaptan restoring systems and exist: Trx and gsh-glutaredoxin approach.Trx and glutaredoxin are used for keeping reduced state by thioredoxin reductase (TrxB) and gsh respectively.Gsh is synthetic by gshA and gshB gene product.Enzyme gsh oxydo-reductase is the gor gene product, need be used for reduction-oxidation type gsh and the catalytic cycle of finishing gsh-glutaredoxin system.

In the strain of trxB null mutation, when normal secretory protein is expressed in tenuigenin and when not having signal sequence, stable disulfide linkage can form in normal secretory protein, as alkaline phosphatase.The also egg oxidation and be used to form disulfide linkage in the trxB mutant strain of two kinds of sulphur oxygen as catalyzer.Find disulfide linkage formation even more effective in the double-mutant strain of disappearance Trx (trxB) and gsh (gor or gshA) approach.Double-mutant strain trxB gor or trxBgshA growth when lacking external source reductive agent such as DTT very slow (time double ~ 300 minutes) also accumulates mutation inhibiting in alkyl hydroperoxidase (ahpC) gene.Gained ahpC* allelotrope is (non-reduced substratum) effectively growth normally, forms disulfide linkage in the tenuigenin and be not included in.Therefore, trxB, gor ahpC* mutant strain (as intestinal bacteria DR473 or FA113) show in the sustenticular cell matter ability that forms disulfide linkage and also can be the same vegetatively with corresponding wild type strain DHB4 good in abundant and minimum medium.

In the present example, alkaline phosphatase activities level that find to express the DHB4 cell performance of TorA-PhoA almost can not detect and the DR473 cell of expressing TorA-PhoA shows very high PhoA activity level.Fractional separation experimental results show that the PhoA activity of surveying up to 50% is because pericentral siphon gathers in the cell lysate.

Because the main catalyzer that disulfide linkage forms is periplasm protein DsbA, secondly the mercaptan in the newly synthetic and transposition albumen of DsbA oxidation determine whether disulfide linkage forms and the complete pericentral siphon that is secreted in tenuigenin.TorA-PhoA is structured among the intestinal bacteria dsbA mutant strain DR473dsbA::kam and expresses.Relatively the PhoA of the homogenic relatively DR473 parental strain of dsbA mutant strain is active shows that activity level much at one in the intact cell lysate.This result proves that the proteic oxidation of PhoA finishes in tenuigenin, along with albumen imports periplasmic space from tenuigenin, stable disulfide linkage can be across inner membrance (table 9).

For measuring the compatible required folding degree of substrate, the eucaryon model protein of test disulfide linkage pattern more sophisticated with the TAT Secretory Pathway.The TorA leading peptide merges the tissue plasminogen activator (vtPA) of clipped form, vtPA is by kringle 2 and have altogether the proteolytic enzyme structural domain (TorA-vtPA) of 9 disulfide linkage form, perhaps merge the heterodimer 2610 anti digoxin antibody fragments that 5 disulfide linkage are arranged, disulfide linkage comprises interchain disulfide linkage (TorA-Fab).Express the DHB4 cell of the identical structure of DR473 cell relative expression of TorA-vtPA and TorA-Fab, in the cell lysate of each expressing protein, show remarkable high-caliber activity.The activity of DHB4 lysate almost can not detect in all situations, except vtPA.The fractional separation experiment further major portion (30-50%) of each albumen gross activity of proof is found in the pericentral siphon part.

In a word, these results show that the continuous proteinic effective secretion of disulfide linkage can take place through the Tat approach, but only these protein foldings are become in the host cell of their native conformations.The active tpA of low background level in the DHB4 cell pericentral siphon shows that this protein at least can be partially folded in reduction tenuigenin.The folded protein of a plurality of disulfide linkage of gained tool is secreted into pericentral siphon as together active-(alkaline phosphatase) or heterodimer (2610 antibody fragment) subsequently.

Embodiment 9

Prove that many disulfide bond proteins are by the output of bacterium double arginine transporter

Detect to determine that disulfide linkage in the trxB gor ahpC mutant strain tenuigenin forms whether be enough to produce can be through the protein of Tat approach output.This is also like this for two kinds of pattern albumen, the PhoA of promptly anti-digoxin (Fab) and Fab fragment.PhoA is made up of two polypeptide chains, always have two disulfide linkage be used for folding and enzymic activity required, and Fab comprises two different chains (two intermolecular disulfide linkage are respectively arranged) that linked to each other by intermolecular disulfide bond.Usually, being formed on of disulfide linkage taken place after outputing to the periplasmic space well-oxygenated environment in these protein.Yet, in analysis the albumen mass-energy of proof tool a plurality of disulfide linkage through the output of Tat system, before they at first in oxidation tenuigenin the folding and further machinery of transporter need the suitably folding pericentral siphon location that is used for of substrate.

A. process

Bacterial isolates, growth and inductive condition:

Used bacterial isolates and plasmid are described in table 7.Bacterial strain DHBA and DRA by will from the dsbA::kan1 allelotrope (MC1000 phoR zih12::Tn10dsbA::kan) of JCB571 respectively transduce coli strain DHB4 and DR473 of P1 obtain.Bacterial strain Coli strain DR473 obtains by transduceing from tatB::kan allelotrope (MC4100 tatB::kan) P1 of MCMTA.Bacterial strain FUDDY obtains by the coli strain FA113 that will transduce from tatC::spec allelotrope (MC4100 tatC::spec) P1 of FUDDY.Coli strain Xl1-Blue (recA1 endA1 gyrA96 thi-1hsdR17 supE44 relA1Lac[F ' proAB lacl ^qZ Δ M15Tn10 (Tet ^г)]) be used for cloning and plasmid propagation.Measure for Phosphoric acid esterase, cell goes down to posterity from overnight culture and cultivates basic M9 substratum [M9 salt, 1 μ g/ml VITMAIN B1, the 1mM MgSO that 0.2% glucose is arranged of 100 times of dilutions ₄, 50 μ g/ml 18 seed amino acids (except methionine(Met) and halfcystine)], 37 ℃ of cultivations subsequently.For Fab research, cell goes down to posterity from overnight culture and cultivates fresh LB substratum (5%v/v), 30 ℃ of cultivations subsequently.Grow into middle exponential phase (OD ₆₀₀~ 0.5), alkaline phosphatase and Fab induce and follow IPTG to add to the final concentration of 0.1mM.The DsbC coexpression is induced with 0.2% pectinose.It is underlined that microbiotic selects to keep the institute that is used on the plasmid with following concentration: ampicillin, 100 μ g/ml; Spectinomycin, 100 μ g/ml and paraxin, 25 μ g ml.

Plasmid construction:

Make up plasmid p33RR by from bacillus coli gene group DNA pcr amplification intestinal bacteria torA signal sequences (ssTorA), use above-mentioned primer TorASacI and TorAXbaI.The DNA of amplification digests and inserts the same loci of pBAD33 with SacI and XbaI.Plasmid p33KK produces identical with p33RR, except mutant primer TorAk (5 '-gcgatggagctcttaaagaggagaaaggtcatgaacaataacgatctctttcaggc atcaaagaaacgttttctggcacaactc-3 ') (SEQ ID NO:129) is used for pcr amplification torA signal sequence.The DNA (PhoA Δ 2-22) of coding no signal sequence phoA passes through to produce from bacillus coli gene group DNA pcr amplification, use primer Phofor (5 '-gcgatgtctagacggacaccagaaatgcctgt-3 ') and (SEQ ID NO:130) and Phorev (5 '-gcgatgaagcttttatttcagccccagagcggctt-3 ') (SEQ ID NO:131).The phoADNA of amplification produces plasmid p33RRP and p33KKP respectively with the same loci of XbaI and HindIII digestion and insertion p33RR and p33KK.Coding torA signal sequence (or torA (R11K; R12K) dna fragmentation frame endomixis phoA signal sequence) uses primer TorASacI (or TorAKK) and Phorev to increase from plasmid p33RRP (or p33KKP).The DNA of pcr amplification produces plasmid pRRP (or pKKP) with the NcoI-HindIII site of BspHI and HindIII digestion and insertion pTrc99.The fusion that makes up alkaline phosphatase and other signal sequence (as ssFdnG, ssFdoG) is as identical carrying out as described in the pTorA-AP.Make up plasmid pTorA-Fab by pcr amplification pTrc99-Fab (Levy etc., 2001) the anti-digoxin two cistron Fab genes of coding in, use primers F abfor (5 '-gctgctagcgaagttcaactgcaacag-3 ') and (SEQ ID NO:132) and Fabrev (5 '-gcgatgcccgggggctttgttagcagccggatctca-3 ') (SEQ ID NO:133), amplification torA signal sequence primer TorASacI and TorAover (5 '-gcgctgttgcagttgaacttcgcta gcagcgtcagtcgccgcttg-3 ') (SEQ ID NO:134).Two kinds of PCR products merge with primer TorASacI and Fabrev through overlapping extension PCR.Overlapping product digests and inserts NcoI and the XmaI site of pTrc99A with BspHI and XmaI.All plasmids are confirmed by order-checking.

Cell grade separates:

Periplasm protein partly obtains (Sargent etc., 1998) by the ice osmotic shock.Cell is specific by centrifugal collection and be resuspended to damping fluid, and damping fluid contains 30mM Tris-HCl (pH8.0), 0.5M sucrose and 1mM Na-EDTA, uses the 20mM iodo-acid amide to prevent that alkaline phosphatase from activating simultaneously.Cell was hatched 1 hour at 25 ℃, and is then centrifugal 10 minutes at 4 ℃ with 5000xg.Precipitation is resuspended to 5ml ice MgSO subsequently ₄And kept on ice 10 minutes.Cell such as preceding centrifugal is collected the supernatant that contains periplasm protein and is used for electrophoretic analysis.Pellet resuspended in 10mlTE (10mM Tris-C1[pH7.5], 2.5mM Na-EDTA) and 20mM iodo-acid amide and French press cell with 2,000lb/in ²Stir evenly.For analyzing the gross protein of untreated cell, directly be resuspended to 10ml TE and 20mM iodo-acid amide, then carry out French press homogenizing.

Enzyme assay:

Expressing the cell of alkaline phosphatase was induced 6 hours.Collect sample, handle and centrifugation with the 20mM iodo-acid amide.The cell of collecting is fractional separation as mentioned above.Soluble protein is quantitative by the Bio-Rad protein determination, uses BSA as standard.Alkaline phosphatase activities is measured as mentioned above.Briefly, equal protein matter is with 200 μ l p-oil of mirbane Phosphoric acid esterase (pNPP; Sigma) solution (1 quick tablet (fast tablet) is at 100ml TrisHCl, among the pH7.4) is hatched, and measures Δ A ₄₀₅To determine each sample neutral and alkali Phosphoric acid esterase hydrolysis rate.Grade efficiency of separation is with serve as a mark enzyme monitoring and measuring as mentioned above of beta-galactosidase enzymes.The correct localized partial data of evaluation of markers enzymic activity 〉=95% only.

ELISA：

Measure following carrying out.96 hole height in conjunction with assay plate (Corning-Costar) with 4 μ g ml ^-1BSA-digoxin conjugate or 4 μ g ml ^-1BSA (100 μ l/ hole) covers (100 μ l/ hole).The plate that covers with the skim-milk among the 5%PBS 4 ℃ of sealings of spending the night.The existence that detects anti-digoxin scFv and Fab antibody is with the anti-mouse IgG of rabbit of dilution in 1: 2000 (be specific to (Fab ') ₂Light chain), the goat anti-rabbit igg of then puting together (H+L) with dilution in 1: 1000 and horseradish peroxidase.Add OPD substrate (Sigma) development and reaction by adding 4.5N H ₂SO ₄Cancellation.Dull and stereotyped on Bio-Tek instrument microtest plate reader in the 490nm reading.

Western blot analysis:

Western blotting is according to (2001) such as Chen.Use following initial antibody: 1: 5, the rabbit alkali-resistivity Phosphoric acid esterase (Rockland), 1: 5 of 000 dilution, the anti-tPA of rabbit of 000 dilution, 1: 5, the anti-mouse IgG of rabbit of 000 dilution (be specific to (Fab ') ₂Light chain, Pierce), 1: 10, the anti-DsbA of mono-clonal rabbit of 000 dilution and anti-DsbC (JohnJoly, Genentech gives) and 1: 10, the mono-clonal rabbit of 000 dilution resists-GroEL (Sigma).Secondary antibodies is 1: 10, goat anti-mouse-HRP of 000 and goat antirabbit-HRP.Film is at first surveyed with initial antibody, and slough in the TBS/2%/SDS/0.7M beta-mercaptoethanol back of developing.The film of wash-out heavily seals and surveys with anti-DsbC and anti-GroEL antibody.

B. many disulfide bond proteins depend on the output policy of TAT in the intestinal bacteria

In bacterium, the oxidation of secretory protein is folding by pericentral siphon enzyme DsbA catalysis, and DsbA is by conformity membrane protein D sbB recirculation.On the contrary, Trx and glutaredoxin approach make tenuigenin be maintained the height reducing environment, are unfavorable for the halfcystine oxidation in the protein.Owing to this reason, need the host protein of disulfide linkage to output to the pericentral siphon compartment, this process is almost specially promoted by the Sec approach in the intestinal bacteria.Exporting this protein by the Tat approach has problem, because the Tat approach is accepted folding albumen usually as substrate.Can not fold in tenuigenin owing to contain the protein of native state disulfide linkage, these protein probably can not be accepted as the substrate of Tat output.Really, some studies have shown that in early days the protein that needs disulfide linkage to be used to fold exports without the Tat approach.

The front is established and proof PhoA merges Trimethylamine 99-N-oxide compound reductase enzyme (TorA) leading peptide or other particular preamble peptide, and the alkaline phosphatase activities of generation can be ignored, and illustrates to lack output.Therefore, infer that suitable folding can be through the output of Tat approach in tenuigenin before the output, the suitably folding formation disulfide linkage that comprises.For this is analyzed, the TorA signal sequence merges escherichia coli alkaline phosphatase (AP) the N-end that lacks its natural signals sequence.Wild-type e. coli cell (DHB4) has plasmid pTorA-AP and induces with IPTG (0.1mM), produces a large amount of tenuigenin A P, detects as western blotting.Yet, do not have detectable AP in the DHB4 cell pericentral siphon part.The activity measurement of identical pericentral siphon part determines to lack the outer AP of kytoplasm.As expected, the AP activity of DHB4 cell matter part is inactivation almost completely, because it can not obtain disulfide linkage in the tenuigenin of this bacterial strain.For whether determining the AP state of oxidation for the output key that depends on TAT, trxB gor aphC intestinal bacteria three mutant strains (bacterial strain DR473) are used to express the ssTorA-AP fusion rotein.When ssTorA-AP expresses from plasmid pTorA-AP (0.1mM IPTG), find that about 25% total enzyme activity is in periplasmic space in bacterial strain DR473.Western blotting is confirmed to take place AP and is separated.The amount that should point out AP in the DR473 cell matter is significantly higher than the DHB4 cell, shows that the AP of malfolding is more vulnerable to the cytoplasm protein cracking.

For supporting this viewpoint, be reported in when lacking one or two disulfide linkage, born of the same parents' internal stability of alkaline phosphatase reduces.Importantly, measure the beta-galactosidase enzymes (LacZ) active (on seeing) of subcellular fraction and the sample that this paper only analyzes LacZ activity＜5% in the pericentral siphon.As second contrast, the cross reaction of tenuigenin companion GroEL and specific corrosioning anteserum is as the contrast of subcellular fraction.On the whole, obviously the folded state of AP is the main determining factor of this protein through Tat approach fan-out capability.

C.PhoA output is that Tat-is special

Recently observe in the situation of some Tat signals, AP can not depend on that the mode of Tat exports.Therefore, be specific to the Tat approach for confirming the AP output among the DR473, the mutant strain of disappearance TorA signal peptide and the AP frame endomixis of no signal sequence are to produce plasmid pKK-AP, and R11 and R12 arginine residues replace (R11K with Methionin in the mutant strain; R12K).Two conservative arginine replace effectively removal transposition (Cristobal etc., 1999) with a pair of Methionin in the document record Tat consensus motif (S/T-R-R-x-F-L-K).As expected, express ssTorA (R11K; R12K)-DHB4 and the DR473 cell of AP fusion rotein can not gather pericentral siphon AP.Importantly, the amount of tenuigenin A P is similar in the DR473 cell, no matter whether RR or KK are present in the leading peptide.Notice ssTorA (R11K in the DHB4 cell matter; R12K)-degree of gathering of the AP ssTorA-AP in the same cell.Possible explanation is that suitable Tat signal (arginine-arginine) makes even the AP target endo cell matter side of malfolding.Thereupon, the film location makes the enzyme of some malfoldings avoid protein cleavage.On the contrary, defective arginine-arginine leading peptide can not suitably interact with Tat mechanism, and the result not AP of target is more vulnerable to the cytoplasm protein cracking.In the plant thylakoid, observe similar phenomenon, wherein big avidin-can be used for the initial identification of film combination and transport mechanism in conjunction with the terminal presequence of the N-of precursor is incorrect substrate of conformation and thereby input failure but adhere to avidin explanation precursor.Therefore, Muser and Theg suggestion Δ pH/Tat correction mechanism must be carried out after precursor identification, but before the transportation committed step.

Depend on that Tat, DR473 use the tatB::kan allelotrope from bacterial strain MCMTA to carry out the P1 transduction to produce bacterial strain owing to determine output separately

(DR473 tatB::kan).As expected,

Cell expressing is from the ssTorA-AP fusion rotein of pTorA-AP, and this cell can not gather AP in pericentral siphon, proves as western blotting and subcellular fraction activity measurement.In addition, when merging two kinds of unlike signal sequences, AP exports in the mode that depends on Tat, and the unlike signal sequence is from hydrogenlyase-N (FDH-N) subunit G (ssFdnG) and FDH-O subunit G (ssFdoG).These results all prove the appearance of AP in the pericentral siphon depend on fully through the output of Tat approach and easily potential energy finish by some different Tat leading peptides.

D. fold with oxidation before the output and in tenuigenin, take place

For whether PhoA oxidation before determining output takes place in tenuigenin, the ssTorA-AP fusion rotein generates in intestinal bacteria dsbA null mutation strain (bacterial strain DHBA and DRA).DsbA is main pericentral siphon enzyme, participates in the formation of disulfide linkage in the normocrinic new synthetic protein of catalysis Sec approach.As a result, because null mutation dsbA, DHBA and DRA mutant strain fully can not the oxidation periplasm proteins.Unexpected is that the pericentral siphon AP that produces from plasmid pTorA-AP (0.1mM IPTG) expression ssTorA-AP in bacterial strain DRA gathers with activity and obtains much at one with DR473 dsbA+ bacterial strain.Therefore, gathering of active A P almost completely is because the AP output of folding and oxidation in tenuigenin in the pericentral siphon compartment.

For determining that this phenomenon is specific to the general feature of TorA presequence or Tat output system, analyze 10 kinds of known and infer Tat leading peptides (table 8).The AP of 10 kinds of signal sequence frame endomixis no signal sequences expresses and analyzes pericentral siphon AP activity 6 kinds of differences but in the background of genetic correlation.Express in bacterial strain DHA (DHB4 dsbA::kan) for determining the active baseline of residue pericentral siphon AP, making up all.Because the AP oxidation all is under an embargo in the tenuigenin of this bacterial strain and pericentral siphon, finds that total AP activity of measuring among the DHA merges for all leading peptides-AP and can ignore (table 9).Remain the pericentral siphon AP active radicals baseline values stdn in view of the above of measuring in 5 kinds of bacterial strains.For relatively, measure AP (Δ 2-22) amount and the discovery of the no signal sequence of exporting in the identical bacterial strain and in all 6 backgrounds, can ignore.

Secondly, express structure in wild-type cell (DHB4) and produce two kinds of Different Results: 1) when tenuigenin was reduction, Tat leader AmiA, FdnG, FdoG, HyaA, HybA and TorA can not export AP; Yet 2) some other Tat leading peptides (DmsA, SufI, YacK and YcbK) can instruct AP to pericentral siphon, even disulfide linkage can not form in tenuigenin.This may be owing to depend on the AP output of Sec.As expected, nearly all leading Toplink instructs the pericentral siphon of AP to bacterial strain DR473, and part is because its tenuigenin of oxidation more.Noticeable exception is ssAmiA and ssHybA, and they can not make AP accumulate in the pericentral siphon of all test strain.Compare the AP activity that the relative DRA of DR473 (DR473 dsbA::kan) finds in pericentral siphon, determine in ssFdnG, ssFdoG, ssHyaA and ssTorA situation, take place folding and oxidation are only finished in AP output in tenuigenin after.Oxidation tenuigenin is arranged but the bacterial strain of disappearance Tat approach (

And DUDDY) expresses in to make up and prove that ssFdnG, ssFdoG and ssTorA instruct AP to pericentral siphon in the special mode of Tat-.On the contrary, the AP output of being instructed by ssSufI, ssYacK and ssYcbK still can take place in tatB and tatC mutant strain, confirms early stage DHB4 result and therefore can use the Sec approach.What is interesting is, at the tatC mutant strain but in the tatB bacterial strain, do not hindered, illustrate that in this leading peptide-AP fusion situation Tat output can not have the generation of TatB albumen by ssHyaA output AP.Should point out when merging ssTorA, observe ColV output similarly and take place with tatC dependence, not dependence of tatB-mode.The subcellular fraction character that all samples carries out is reported in table 9, confirms by LacZ activity measurement and protein spots trace.

At last, for the situation that ssFdnG-AP expresses, carry out the western blot analysis and the AP activity measurement (Fig. 7) of pericentral siphon and tenuigenin part in all 6 kinds of genetic backgrounds.Notice the AP amount measured in total AP activity (pericentral siphon and tenuigenin) of finding among the DR473/pFdnG-AP and the DR473 tenuigenin much at one, DR473 expresses the AP from the no signal sequence form of plasmid pAID135.From then on obvious in the situation of ssFdnG leading peptide in the data, AP must folding and oxidation before by the transposition of Tat mechanism.According to inventor's knowledge, this is to prove the disulfide linkage stable maintenance in the film transposition of depending on Tat that from the beginning forms in the tenuigenin first.PhoA be as monomer (~ 48kDa) with its active homodimer state (~ 96kDa) transposition is still unclear, although known PhoA is folded into its high stability natural dimer state rapidly.In addition, the big alkaline phosphatase dimer viewpoint compatible with Tat mechanism studies have shown that by the research support of front the 142kDa FdnGH Asia complex body of intestinal bacteria hydrogenlyase-N is transported by the Tat system.

Embodiment 10

Tat-regulates folding anti digoxin antibody fragment ' hitchhiking ' strategy from the output of Bacillus coli cells matter

Quite a few protein by the output of Tat approach is enzyme, and enzyme obtains the cofactor in the tenuigenin and generally brings into play function (as intestinal bacteria Trimethylamine 99-N-oxide compound reductase enzyme) in breathing or electron transfer process before output.The cofactor that obtains in the tenuigenin needs only roughly complete in the tertiary structure that folds the back generation.Along with these are arranged, find to obtain to depend primarily on small subunit HybO output by film target and the nickel of HybC, HybC is the big subunit of intestinal bacteria hydrogenase 2, HybO comprises the special leading peptide of Tat-.Optimization model is that the little and big subunit of hydrogenase 2 at first forms complex body in tenuigenin, and complex body passes through the leading peptide target film of small subunit subsequently.Whether the complex body of natural generation is similar therewith, test when non-physiological heterodimer antibody fragment suitably folds in tenuigenin and can export through the Tat transporter.Surprisingly, discovery Tat approach also can be exported the heterodimer that disulfide linkage links to each other, and wherein only a polypeptide chain merges the TorA leading peptide (referring to chimeric, Fig. 6).

Use is specific to the Fab antibody fragment of cardiac glycoside digoxin, forms with light chain by two polypeptide chains are promptly heavy, links to each other through disulfide linkage.In addition, heavy and light chain respectively comprises two intramolecular disulfide bonds.The TorA leading peptide only merges heavy chain (V _H-C _H1), heavy chain with from the light chain (V of bicistronic mRNA ₁-C ₁) coexpression.By this mode, the TorA-heavy chain carries light chain to pericentral siphon with " bearing " formula, and this only takes place when the interchain disulphide bridges at first is formed in the tenuigenin before transposition.

Oxidation tenuigenin (bacterial strain DRA) is being arranged and lacking in the mutant strain of dsbA, complete Fab albumen is by the output of Tat approach, but only sub-fraction Fab location (~ 15-20%) in the osmotic shock part, confirm as western blotting.The folding output of anti-digoxin Fab significantly increases in the earlier report tenuigenin, and this is pericentral siphon disulphide isomerase DsbC (Δ ssDsbC) or GroEL by coexpression no signal sequence form.In this analysis, coexpression Δ ssDsbC causes that the amount of Fab significantly increases in the pericentral siphon (~ 50% osmotic shock part).This may probably improve folded protein output because of it because the protein mass that the coexpression mate molecule enables to export in the tenuigenin increases.

Fab surveys with the initial antibody immunity of identification mouse sequence of light chain.Therefore, seen band proof light chain forms by intramolecular disulfide bond suitably to be collected by heavy chain, is delivered to periplasmic space subsequently.The location proof subcellular fractionation of tenuigenin labelled protein GroEL and pericentral siphon labelled protein DsbC separates successfully.The Fab albumen of DRA cell pericentral siphon part is correctly folding and function arranged, and proves as the ability of its conjugated antigen digoxin in ELISA measures.

When the RR of TorA leader dipeptides was mutated into KK or having in the cytoplasmic DHB4 cell of reduction, owing to merge with ssTorA-AP, Fab was removed fully appearing in the tatB mutant strain of osmotic shock part.In addition, when hatching under increasing the infiltrative condition of adventitia (Chen etc., 2001), the cell of finishing of expressing Fab antibody outputs to pericentral siphon through the Tat approach, and cell can be used fluorescent antigen digoxin-bodipy specific mark.The fluorescence of these cells than DHA or

Observed background fluorescence is high 5 times in the control cells.These are general description as a result: (i) the Tat approach can stride film export the Fab of abundant oxidation and (ii) this process depend on intracytoplasmic intermolecular disulfide bond formation before the set of light and heavy chain and the output.Transhipment oxidation Fab molecule is to pericentral siphon, it may be abundant oxidation, the conclusive evidence pattern of hitchhiking that proof hints previously is provided, wherein contains the polypeptide transposition of second kind of no leader of polypeptides for modulating of Tat leading peptide, the polypeptide that contains leading peptide links to each other with tenuigenin by second peptide species.

This institute of table 7. bacterial isolates and plasmid

Table 8: the leading peptide aminoacid sequence that can rely on Tat output alkaline phosphatase

AmiA ^＊ MSTFKPLKTLT SRRQVLKAGLAALTLSGMSQAIAK(SEQ?IDNO：33)

DmsA MKTKIPDAVLAAEV SRRGLVKTTAIGGLAMASSALTLPFS

IAHAV(SEQID?NO：43)

FdnG MDV SRRQFFKICAGGMAGTTVAALGFAP

QALAQ(SEQID?NO：127)

FdoG MQV SRRQFFKICAGGMAGTTAAALGFAPSVALAE(SEQID?NO：45)

HyaA ^＊ MNNEETFYQAMRRQGV TRRSFLKYCSLAATSLGLGAGMAP

IAWAL(SEQID?NO：28)

HybA M NRRNFIKAASCGALLTGALPSVSHAAA(SEQ?IDNO：36)

SufI MSL SRRQFIQASGIALCAGAVPL

ASAA(SEQ?IDNO：38)

TorA MNNNDLFQA SRRRFLAQLGGLTVAGMLGPSLLTP

ATAA(SEQID?NO：128)

YacK M QRRDFLKYSVALGVASALPLWS

AVFAA(SEQ?IDNO：29)

YcbK MDKFDA NRRKLLALGGVALGAAILPTPAFAT(SEQ?IDNO：30)

Table 9. obtains to merge at the escherichia coli alkaline phosphatase of inferring intestinal bacteria Tat signal peptide and no leader from pericentral siphon alkaline phosphatase (AP) activity that merges ^*Between

^*Relative alkaline phosphatase activities is by calculating according to the active stdn of surveying in the DHA control strain active in the sample.The report value of alkaline phosphatase activities is the mean value from 3 independent measurements of 2 independent experiments (n=6).For all report values, standard deviation is less than 10%.The substantial activity of being surveyed in the value representation DHA control strain in the bracket.

^aThe AP of no signal sequence makes up

^bAccording to surveying active standardized value among the DHA/ssHyaA-AP

^cCarry the signal sequence of c-zone positive charge

Nd=can not detect

* AmiA and HyaA are contrast Tat leading peptides.They all can not export alkaline phosphatase under this paper study condition.

Reference

Following reference provides demonstration program or other details to replenish content shown in this paper, and it is for reference to include this paper especially in.

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Sequence table

＜110〉G. George Europe (GEORGIOU, GEORGE)

M. De Lisha (DELISA, MATTHEW)

＜120〉leading peptide of structure secreting bacteria recombinant protein

<130>CLFR：019CN

<140>PCT/US02/35618

<141>2002-11-05

<150>60/337,452

<151>2001-11-05

<160>134

<170>PatentIn?Ver.2.1

<210>1

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(3)

＜223〉Xaa=is any

<400>1

Arg?Arg?Xaa?Phe?Leu?Lys

1 5

<210>2

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>2

Ser?Arg?Arg?Arg?Phe?Leu?Lys

1 5

<210>3

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(4)..(7)

＜223〉Xaa=is any

<400>3

Ser?Arg?Arg?Xaa?Phe?Leu?Xaa

1 5

<210>4

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(4)..(7)

＜223〉Xaa=is any

<400>4

Thr?Arg?Arg?Xaa?Phe?Leu?Xaa

1 5

<210>5

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(4)..(5)

＜223〉Xaa=is any

<400>5

Ser?Arg?Arg?Xaa?Xaa?Leu?Lys

1 5

<210>6

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(4)..(5)

＜223〉Xaa=is any

<400>6

Ser?Arg?Arg?Xaa?Xaa?Leu?Ala

1 5

<210>7

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(4)..(5)

＜223〉Xaa=is any

<400>7

Thr?Arg?Arg?Xaa?Xaa?Leu?Lys

1 5

<210>8

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(4)..(5)

＜223〉Xaa=is any

<400>8

Thr?Arg?Arg?Xaa?Xaa?Leu?Ala

1 5

<210>9

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(4)..(5)

＜223〉Xaa=is any

<400>9

Ser?Arg?Arg?Xaa?Xaa?Leu?Thr

1 5

<210>10

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(4)..(5)

＜223〉Xaa=is any

<400>10

Ser?Arg?Arg?Xaa?Xaa?Ile?Lys

1 5

<210>11

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(4)..(5)

＜223〉Xaa=is any

<400>11

Ser?Arg?Arg?Xaa?Xaa?Ile?Ala

1 5

<210>12

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(4)..(7)

＜223〉Xaa=is any

<400>12

Ser?Arg?Arg?Xaa?Phe?Ile?Xaa

1 5

<210>13

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(4)

＜223〉Xaa=is any

<400>13

Ser?Arg?Arg?Xaa?Phe?Met?Lys

1 5

<210>14

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(4)

＜223〉Xaa=is any

<400>14

Ser?Arg?Arg?Xaa?Phe?Val?Lys

1 5

<210>15

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(4)

＜223〉Xaa=is any

<400>15

Ser?Arg?Arg?Xaa?Phe?Val?Ala

1 5

<210>16

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>16

Ser?Arg?Arg?Gln?Phe?Leu?Lys

1 5

<210>17

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(3)

＜223〉Xaa=is any

<400>17

Arg?Arg?Xaa?Phe?Leu?Ala

1 5

<210>18

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(3)

＜223〉Xaa=is any

<400>18

Arg?Arg?Xaa?Phe?Leu?Lys

1 5

<210>19

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>19

gcgatggagc?tc?ttaaagag?gagaaaggtc 30

<210>20

<211>12

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>20

gcgatgtcta?ga 12

<210>21

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(4)

＜223〉Xaa=is any

<400>21

Ser?Arg?Arg?Xaa?Phe?Met?Lys

1 5

<210>22

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(4)

＜223〉Xaa=is any

<400>22

Ser?Arg?Arg?Xaa?Phe?Val?Lys

1 5

<210>23

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<221>MOD-RES

<222>(4)

＜223〉Xaa=is any

<400>23

Ser?Arg?Arg?Xaa?Phe?Val?Ala

1 5

<210>24

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>24

Ser?Arg?Arg?Gln?Phe?Leu?Lys

1 5

<210>25

<211>29

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>25

Met?Pro?Phe?Lys?Lys?Leu?Ser?Arg?Arg?Thr?Phe?Leu?Thr?Ala?Ser?Ser

1 5 10 15

Ala?Leu?Ala?Phe?Leu?His?Thr?Pro?Phe?Ala?Arg?Ala?Leu

20 25

<210>26

<211>28

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>26

Met?Thr?Trp?Ser?Arg?Arg?Gln?Phe?Leu?Thr?Gly?Val?Gly?Val?Leu?Ala

1 5 10 15

Ala?Val?Ser?Gly?Thr?Ala?Gly?Arg?Val?Val?Ala?Lys

20 25

<210>27

<211>34

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>27

Met?Lys?Glu?Ser?Asn?Ser?Arg?Arg?Glu?Phe?Leu?Ser?Gln?Ser?Gly?Lys

1 5 10 15

Met?Val?Thr?Ala?Ala?Ala?Leu?Phe?Gly?Thr?Ser?Val?Pro?Leu?Ala?His

20 25 30

Ala?Ala

<210>28

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>28

Met?Asn?Asn?Glu?Glu?Thr?Phe?Tyr?Gln?Ala?Met?Arg?Arg?Gln?Gly?Val

1 5 10 15

Thr?Arg?Arg?Ser?Phe?Leu?Lys?Tyr?Cys?Ser?Leu?Ala?Ala?Thr?Ser?Leu

20 25 30

Gly?Leu?Gly?Ala?Gly?Met?Ala?Pro?Lys?Ile?Ala?Trp?Ala?Leu

35 40 45

<210>29

<211>29

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>29

Met?Gln?Arg?Arg?Asp?Phe?Leu?Lys?Tyr?Ser?Val?Ala?Leu?Gly?Val?Ala

1 5 10 15

Ser?Ala?Leu?Pro?Leu?Trp?Ser?Arg?Ala?Val?Phe?Ala?Ala

20 25

<210>30

<211>31

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>30

Met?Asp?Lys?Phe?Asp?Ala?Asn?Arg?Arg?Lys?Leu?Leu?Ala?Leu?Gly?Gly

1 5 10 15

Val?Ala?Leu?Gly?Ala?Ala?Ile?Leu?Pro?Thr?Pro?Ala?Phe?Ala?Thr

20 25 30

<210>31

<211>32

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>31

Met?Arg?His?Ile?Phe?Gln?Arg?Leu?Leu?Pro?Arg?Arg?Leu?Trp?Leu?Ala

1 5 10 15

Gly?Leu?Pro?Cys?Leu?Ala?Leu?Leu?Gly?Cys?Val?Gln?Asn?His?Asn?Lys

20 25 30

<210>32

<211>36

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>32

Met?Gln?Tyr?Lys?Asp?Glu?Asn?Gly?Val?Asn?Glu?Pro?Ser?Arg?Arg?Arg

1 5 10 15

Leu?Leu?Lys?Val?Ile?Gly?Ala?Leu?Ala?Leu?Ala?Gly?Ser?Cys?Pro?Val

20 25 30

Ala?His?Ala?Gln

35

<210>33

<211>35

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>33

Met?Ser?Thr?Phe?Lys?Pro?Leu?Lys?Thr?Leu?Thr?Ser?Arg?Arg?Gln?Val

1 5 10 15

Leu?Lys?Ala?Gly?Leu?Ala?Ala?Leu?Thr?Leu?Ser?Gly?Met?Ser?Gln?Ala

20 25 30

Ile?Ala?Lys

35

<210>34

<211>45

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>34

Met?Lys?Lys?Asn?Gln?Phe?Leu?Lys?Glu?Ser?Asp?Val?Thr?Ala?Glu?Ser

1 5 10 15

Val?Phe?Phe?Met?Lys?Arg?Arg?Gln?Val?Leu?Lys?Ala?Leu?Gly?Ile?Ser

20 25 30

Ala?Thr?Ala?Leu?Ser?Leu?Pro?His?Ala?Ala?His?Ala?Asp

35 40 45

<210>35

<211>31

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>35

Met?Ser?Gly?Leu?Pro?Leu?Ile?Ser?Arg?Arg?Arg?Leu?Leu?Thr?Ala?Met

1 5 10 15

Ala?Leu?Ser?Pro?Leu?Leu?Trp?Gln?Met?Asn?Thr?Ala?His?Ala?Ala

20 25 30

<210>36

<211>28

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>36

Met?Asn?Arg?Arg?Asn?Phe?Ile?Lys?Ala?Ala?Ser?Cys?Gly?Ala?Leu?Leu

1 5 10 15

Thr?Gly?Ala?Leu?Pro?Ser?Val?Ser?His?Ala?Ala?Ala

20 25

<210>37

<211>33

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>37

Met?Asp?Arg?Arg?Arg?Phe?Ile?Lys?Gly?Ser?Met?Ala?Met?Ala?Ala?Val

1 5 10 15

Cys?Gly?Thr?Ser?Gly?Ile?Ala?Ser?Leu?Phe?Ser?Gln?Ala?Ala?Phe?Ala

20 25 30

Ala

<210>38

<211>28

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>38

Met?Ser?Leu?Ser?Arg?Arg?Gln?Phe?Ile?Gln?Ala?Ser?Gly?Ile?Ala?Leu

1 5 10 15

Cys?Ala?Gly?Ala?Val?Pro?Leu?Lys?Ala?Ser?Ala?Ala

20 25

<210>39

<211>49

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>39

Met?Ser?Asn?Gln?Gly?Glu?Tyr?Pro?Glu?Asp?Asn?Arg?Val?Gly?Lys?His

1 5 10 15

Glu?Pro?His?Asp?Leu?Ser?Leu?Thr?Arg?Arg?Asp?Leu?Ile?Lys?Val?Ser

20 25 30

Ala?Ala?Thr?Ala?Ala?Thr?Ala?Val?Val?Tyr?Pro?His?Ser?Thr?Leu?Ala

35 40 45

Ala

<210>40

<211>45

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>40

Met?Ser?Trp?Ile?Gly?Trp?Thr?Val?Ala?Ala?Thr?Ala?Leu?Gly?Asp?Asn

1 5 10 15

Gln?Met?Ser?Phe?Thr?Arg?Arg?Lys?Phe?Val?Leu?Gly?Met?Gly?Thr?Val

20 25 30

Ile?Phe?Phe?Thr?Gly?Ser?Ala?Ser?Ser?Leu?Leu?Ala?Asn

35 40 45

<210>41

<211>38

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>41

Met?Thr?Gly?Asp?Asn?Thr?Leu?Ile?His?Ser?His?Gly?Ile?Asn?Arg?Arg

1 5 10 15

Asp?Phe?Met?Lys?Leu?Cys?Ala?Ala?Leu?Ala?Ala?Thr?Met?Gly?Leu?Ser

20 25 30

Ser?Lys?Ala?Ala?Ala?Glu

35

<210>42

<211>47

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>42

Met?Met?Lys?Ile?His?Thr?Thr?Glu?Ala?Leu?Met?Lys?Ala?Glu?Ile?Ser

1 5 10 15

Arg?Arg?Ser?Leu?Met?Lys?Thr?Ser?Ala?Leu?Gly?Ser?Leu?Ala?Leu?Ala

20 25 30

Ser?Ser?Ala?Phe?Thr?Leu?Pro?Phe?Ser?Gln?Met?Val?Arg?Ala?Ala

35 40 45

<210>43

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>43

Met?Lys?Thr?Lys?Ile?Pro?Asp?Ala?Val?Leu?Ala?Ala?Glu?Val?Ser?Arg

1 5 10 15

Arg?Gly?Leu?Val?Lys?Thr?Thr?Ala?Ile?Gly?Gly?Leu?Ala?Met?Ala?Ser

20 25 30

Ser?Ala?Leu?Thr?Leu?Pro?Phe?Ser?Arg?Ile?Ala?His?Ala?Val

35 40 45

<210>44

<211>44

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>44

Met?Ser?Lys?Asn?Glu?Arg?Met?Val?Gly?Ile?Ser?Arg?Arg?Thr?Leu?Val

1 5 10 15

Lys?Ser?Thr?Ala?Ile?Gly?Ser?Leu?Ala?Leu?Ala?Ala?Gly?Gly?Phe?Ser

20 25 30

Leu?Pro?Phe?Thr?Leu?Arg?Asn?Ala?Ala?Ala?Ala?Val

35 40

<210>45

<211>34

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>45

Met?Gln?Val?Ser?Arg?Arg?Gln?Phe?Phe?Lys?Ile?Cys?Ala?Gly?Gly?Met

1 5 10 15

Ala?Gly?Thr?Thr?Ala?Ala?Ala?Leu?Gly?Phe?Ala?Pro?Ser?Val?Ala?Leu

20 25 30

Ala?Glu

<210>46

<211>62

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>46

Met?Thr?Asp?Tyr?Ala?Ser?Phe?Ala?Lys?Val?Ser?Gly?Gln?Ile?Ser?Arg

1 5 10 15

Leu?Leu?Val?Thr?Gln?Leu?Arg?Phe?Leu?Leu?Leu?Gly?Arg?Gly?Met?Ser

20 25 30

Gly?Ser?Asn?Thr?Ala?Ile?Ser?Arg?Arg?Arg?Leu?Leu?Gln?Gly?Ala?Gly

35 40 45

Ala?Met?Trp?Leu?Leu?Ser?Val?Ser?Gln?Val?Ser?Leu?Ala?Ala

50 55 60

<210>47

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>47

Arg?Arg?Arg?Gly?Phe?Leu?Thr

1 5

<210>48

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>48

Gln?Arg?Arg?Arg?Ala?Leu?Thr

1 5

<210>49

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>49

Thr?Arg?Arg?Glu?Phe?Ile?Lys

1 5

<210>50

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>50

Ser?Arg?Arg?Ser?Phe?Met?Lys

1 5

<210>51

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>51

Gly?Arg?Arg?Arg?Phe?Leu?Arg

1 5

<210>52

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>52

Ser?Arg?Arg?Gln?Phe?Phe?Lys

1 5

<210>53

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>53

Ser?Arg?Arg?Arg?Phe?Leu?Gln

1 5

<210>54

<211>54

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>54

gcgatggagc?tcttaaagag?gagaaaggtc?atgccattta?aaaaactctc?ccga 54

<210>55

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>55

gcgatggagc?tcttaaagag?gagaaaggtc?atgacctggt?ctcgtcgc 48

<210>56

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>56

gcgatggagc?tcttaaagag?gagaaaggtc?atgaaagaaa?gcaatagc 48

<210>57

<211>57

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>57

gcgatggagc?tcttaaagag?gagaaaggtc?atgaataacg?aggaaacatt?ttaccag 57

<210>58

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>58

gcgatggagc?tcttaaagag?gagaaaggtc?gtggggagac?gacgcgga 48

<210>59

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>59

gcgatggagc?tcttaaagag?gagaaaggtc?atgcaacgtc?gtgatttc 48

<210>60

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>60

gcgatggagc?tcttaaagag?gagaaaggtc?atgtcccggt?cagcgaaa 48

<210>61

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>61

gcgatggagc?tcttaaagag?gagaaaggtc?atggacaaat?tcgacgct 48

<210>62

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>62

gcgatggagc?tcttaaagag?gagaaaggtc?atgcgacaca?tttttcaa 48

<210>63

<211>53

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>63

gcgatggagc?tcttaaagag?gagaaaggtc?atgcagtata?aagatgaaaa?cgg 53

<210>64

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>64

gcgatggagc?tcttaaagag?gagaaaggtc?atgagcactt?ttaaacca 48

<210>65

<211>59

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>65

gcgatggagc?tcttaaagag?gagaaaggtc?atgaaaaaga?atcaattttt?aaaagaatc?59

<210>66

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>66

gcgatggagc?tcttaaagag?gagaaaggtc?atgagcggct?tacctctt 48

<210>67

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>67

gcgatggagc?tcttaaagag?gagaaaggtc?atgattcggc?aacgtcgt 48

<210>68

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>68

gcgatggagc?tcttaaagag?gagaaaggtc?atgatcaggg?aggaagtt 48

<210>69

<211>62

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>69

gcgatggagc?tcttaaagag?gagaaaggtc?gtgaacagac?gtaattttat?taaagcagcc?60

tc 62

<210>70

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>70

gcgatggagc?tc?ttaaagag?gagaaaggtc?atggatcgta?gacgattt 48

<210>71

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>71

gcgatggagc?tcttaaagag?gagaaaggtc?atgtcactca?gtcggcgt 48

<210>72

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>72

gcgatggagc?tcttaaagag?gagaaaggtc?atgagcaacc?aaggcgaa 48

<210>73

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>73

gcgatggagc?tcttaaagag?gagaaaggtc?atgtcatgga?tagggtgg 48

<210>74

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>74

gcgatggagc?tcttaaagag?gagaaaggtc?atgactggag?ataacacc 48

<210>75

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>75

gcgatggagc?tcttaaagag?gagaaaggtc?atgaaactca?gtcgtcgt 48

<210>76

<211>57

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>76

gcgatggagc?tcttaaagag?gagaaaggtc?atgatgaaaa?tccataccac?agaggcg 57

<210>77

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>77

gcgatggagc?tcttaaagag?gagaaaggtc?atgaaaacga?aaatccctga?tg 52

<210>78

<211>54

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>78

gcgatggagc?tcttaaagag?gagaaaggtc?atgtccaaaa?atgaacgaat?ggtg 54

<210>79

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>79

gcgatggagc?tcttaaagag?gagaaaggtc?atggacgtca?gtcgcaga 48

<210>80

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>80

gcgatggagc?tcttaaagag?gagaaaggtc?atgcaggtca?gcagaagg 48

<210>81

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>81

gcgatggagc?tcttaaagag?gagaaaggtc?atgacagatt?atgcgtcttt?cgctaaagtt?60

<210>82

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>82

gcgatggagc?tcttaaagag?gagaaaggtc?atgatttcac?gccgccga 48

<210>83

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>83

gcgatgtcta?gagctttgtc?gggcgggaag 30

<210>84

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>84

gcgatgtcta?gaattgatat?tcaacgtttt?cgccac 36

<210>85

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>85

gcgatgtcta?gatagggtgc?cagctaccgc 30

<210>86

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>86

gcgatgtcta?gagcgcggtt?tgttctccag 30

<210>87

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>87

gcgatgtcta?gatacgcgcc?cgatatggtt 30

<210>88

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>88

gcgatgtcta?gataacgttg?ggcgttctgc 30

<210>89

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>89

gcgatgtcta?gagcgcaacc?gcacgccaga 30

<210>90

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>90

gcgatgtcta?gagcgtgggg?tagagagtgt 30

<210>91

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>91

gcgatgtcta?gacgtatcaa?tggctggctt 30

<210>92

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>92

gcgatgtcta?gacgcacttt?gcgttttttg 30

<210>93

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>93

gcgatgtcta?gattttaaaa?gttcgtcttt?gg 32

<210>94

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>94

gcgatgtcta?gaaaaccagc?taagcagatc 30

<210>95

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>95

gcgatgtcta?gaattgggat?caatagccgc 30

<210>96

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>96

gcgatgtcta?gagaatacag?cgaccgtatg 30

<210>97

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>97

gcgatgtcta?gatttaccgc?ccttctcttc 30

<210>98

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>98

gcgatgtcta?gatggcgggc?ggttttcagc 30

<210>99

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>99

gcgatgtcta?gaggcaatat?cagaatctgc 30

<210>100

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>100

gcgatgtcta?gacggttgct?gttgcccggc 30

<210>101

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>101

gcgatgtcta?gaagctgccg?gaacgcttgc 30

<210>102

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>102

gcgatgtcta?gacttttctt?gcctcgtgtt 30

<210>103

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>103

gcgatgtcta?gaaaccgatt?cggccatctc 30

<210>104

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>104

gcgatgtcta?gactgaccaa?caacggcgcg 30

<210>105

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>105

gcgatgtcta?gattctaccg?gagcctctgc 30

<210>106

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>106

gcgatgtcta?gatggaatgg?cgctatcgac 30

<210>107

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>107

gcgatgtcta?gatttttcgc?gggcctgttg 30

<210>108

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>108

gcgatgtcta?gataatttgt?agtttcgcgc?ctg 33

<210>109

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>109

gcgatgtcta?gacagtttat?actgccgggt?ttc 33

<210>110

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>110

gcgatgtcta?gacgccacga?cctggctgac 30

<210>111

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>111

gcgatgtcta?gagctcgtgg?ctatcgtcgc 30

<210>112

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>112

gcgatgtcta?gaagtaaagg?agaagaactt?ttcact?36

<210>113

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>113

gcgatgaagc?ttctatttgt?atagttcatc?cat 33

<210>114

<211>81

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>114

gcgatgaagc?ttgcatgctt?aagctgctaa?agcgtagttt?tcgtcgtttg?ctgcgtcgac 60

tttgtatagt?tcatccatgc?c 81

<210>115

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>115

gcgatggaat?tcgagctctt?aaagaggaga?aaggtcatga?acaataacga?tctctttcag 60

<210>116

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>116

gcgatgtcta?gaagcgtcag?tcgccgcttg?cgccgc 36

<210>117

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>117

gcgatggaat?tcgagctctt?aaagaggaga?aaggtcgtga?aacaaagcac?tattgcactg?60

<210>118

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>118

gcgatgaagc?ttttatttca?gccccagagc?ggctt 35

<210>119

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>119

Ala?Ala?Asn?Asp?Glu?Asn?Tyr?Ala?Leu?Ala?Ala

1 5 10

<210>120

<211>45

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>120

Met?Asn?Asn?Asn?Asp?Leu?Phe?Gln?Ala?Ser?Arg?Arg?Arg?Phe?Leu?Ala

1 5 10 15

Gln?Leu?Gly?Gly?Leu?Thr?Val?Ala?Gly?Met?Leu?Gly?Pro?Ser?Leu?Leu

20 25 30

Thr?Pro?Arg?Arg?Ala?Thr?Ala?Ala?Gln?Ala?Ala?Thr?Asp

35 40 45

<210>121

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide＜400〉121

Met?Asn?Asn?Asn?Asp?Leu?Phe?Gln?Thr?Ser?Arg?Arg?Arg?Leu?Leu?Ala

1 5 10 15

Gln?Leu?Gly?Gly?Leu?Thr?Val?Ala?Gly?Met?Leu?Gly?Pro?Ser?Leu?Leu

20 25 30

Thr?Pro?Arg?Arg?Ala?Thr?Ala?Ala?Gln?Ala?Ala?Thr?Asp?Ala

35 40 45

<210>122

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>122

Met?Asn?Asn?Asn?Asp?Leu?Phe?Gln?Thr?Ser?Arg?Gln?Arg?Phe?Leu?Ala

1 5 10 15

Gln?Leu?Gly?Gly?Leu?Thr?Val?Ala?Gly?Met?Leu?Gly?Pro?Ser?Leu?Leu

20 25 30

Thr?Pro?Arg?Arg?Ala?Thr?Ala?Ala?Gln?Ala?Ala?Thr?Asp?Ala

35 40 45

<210>123

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>123

Met?Asn?Asn?Asn?Asp?Ile?Phe?Gln?Ala?Ser?Arg?Arg?Arg?Phe?Leu?Ala

1 5 10 15

Gln?Pro?Gly?Gly?Leu?Thr?Val?Ala?Gly?Met?Leu?Gly?Pro?Ser?Leu?Leu

20 25 30

Thr?Pro?Arg?Arg?Ala?Thr?Ala?Ala?Gln?Ala?Ala?Thr?Asp?Ala

35 40 45

<210>124

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>124

Met?Asn?Asn?Asn?Glu?Leu?Phe?Gln?Ala?Ser?Arg?Arg?Arg?Phe?Leu?Ala

1 5 10 15

Gln?Leu?Gly?Gly?Leu?Thr?Val?Ala?Gly?Met?Leu?Gly?Pro?Ser?Leu?Leu

20 25 30

Thr?Pro?Arg?Arg?Ala?Thr?Ala?Ala?Gln?Ala?Ala?Thr?Asp?Ala

35 40 45

<210>125

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>125

Met?Asn?Asn?Asn?Asp?Leu?Phe?Gln?Thr?Thr?Arg?Arg?Arg?Phe?Leu?Ala

1 5 10 15

Gln?Leu?Gly?Gly?Leu?Thr?Val?Ala?Gly?Met?Leu?Gly?Pro?Ser?Leu?Leu

20 25 30

Thr?Pro?Arg?Arg?Ala?Thr?Ala?Ala?Gln?Ala?Ala?Thr?Asp?Ala

35 40 45

<210>126

<211>46

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>126

Met?Asn?Asn?Asn?Asp?Ser?Phe?Gln?Thr?Ser?Arg?Arg?Arg?Phe?Leu?Ala

1 5 10 15

Gln?Leu?Gly?Gly?Leu?Thr?Val?Ala?Gly?Met?Leu?Gly?Pro?Ser?Leu?Leu

20 25 30

Thr?Pro?Arg?Arg?Ala?Thr?Ala?Ala?Gln?Ala?Ala?Thr?Asp?Ala

35 40 45

<210>127

<211>34

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>127

Gly?Cys?Gly?Ala?Thr?Gly?Gly?Ala?Gly?Cys?Thr?Cys?Thr?Thr?Ala?Ala

1 5 10 15

Ala?Gly?Ala?Gly?Gly?Ala?Gly?Ala?Ala?Ala?Gly?Gly?Thr?Cys?Ala?Thr

20 25 30

Gly?Ala?Ala?Cys?Ala?Ala?Thr?Ala?Ala?Cys?Gly?Ala?Thr?Cys?Thr?Cys

35 40 45

Thr?Thr?Thr?Cys?Ala?Gly?Gly?Cys?Ala?Thr?Cys?Ala?Ala?Ala?Gly?Ala

50 55 60

Ala?Ala?Cys?Gly?Thr?Thr?Thr?Thr?Cys?Thr?Gly?Gly?Cys?Ala?Cys?Ala

65 70 75 80

Ala?Cys?Thr?Cys

<210>128

<211>40

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>128

Gly?Cys?Gly?Cys?Thr?Gly?Thr?Thr?Gly?Cys?Ala?Gly?Thr?Thr?Gly?Ala

1 5 10 15

Ala?Cys?Thr?Thr?Cys?Gly?Cys?Thr?Ala?Gly?Cys?Ala?Gly?Cys?Gly?Thr

20 25 30

Cys?Ala?Gly?Thr?Cys?Gly?Cys?Cys?Gly?Cys?Thr?Thr?Gly

35 40 45

<210>129

<211>84

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>129

gcgatggagc?tcttaaagag?gagaaaggtc?atgaacaata?acgatctctt?tcaggcatca?60

aagaaacgtt?ttctggcaca?actc 84

<210>130

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>130

gcgatgtcta?gacggacacc?agaaatgcct?gt 32

<210>131

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>131

gcgatgaagc?ttttatttca?gccccagagc?ggctt 35

<210>132

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>132

gctgctagcg?aagt?tcaact?gcaacag 27

<210>133

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>133

gcgatgcccg?ggggctttgt?tagcagccgg?atctca 36

<210>134

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer

<400>134

gcgctgttgc?agttgaactt?cgctagcagc?gtcagtcgcc?gcttg 45

Claims (12)

1. method that generates the biological activity heterologous polypeptide in cell is characterized in that described method comprises step:

A) construction expression box comprises the nucleotide sequence upstream that the nucleotide sequence of coding leading peptide is placed the described biological activity heterologous polypeptide of encoding, and described leading peptide instructs described biological activity heterologous polypeptide to export through double arginine transposition approach; With

B) described expression cassette is expressed in bacterial cell, and wherein said biological activity heterologous polypeptide generates with biologically active form;

Wherein, described bacterial cell has oxidation tenuigenin, and at least a biological activity heterologous polypeptide of described emiocytosis, described heterologous polypeptide contain 2 to 17 disulfide linkage.

2. the method for claim 1 is characterized in that, described biological activity heterologous polypeptide comprises antibody fragment.

3. the method for claim 1 is characterized in that, described leading peptide comprises the sequence that is selected from SEQ IDNOs:25-46 and 120-128.

4. the method for claim 1, it is characterized in that, described biological activity heterologous polypeptide be selected from described bacterial cell excretory polypeptide, can be from the pericentral siphon isolated polypeptide of described bacterial cell, conformity membrane albumen and can be from described bacterial cell culture supernatant isolated polypeptide.

5. the method for claim 1 is characterized in that, described biological activity heterologous polypeptide is the Mammals polypeptide.

6. method as claimed in claim 5 is characterized in that, described Mammals polypeptide is selected from tissue plasminogen activator, pancreatic trypsin inhibitor, antibody, antibody fragment and toxin immunity albumen.

7. the method for claim 1 is characterized in that, described biological activity heterologous polypeptide is selected from polypeptide, mutant polypeptide and the brachymemma polypeptide of native conformation.

8. the method for claim 1 is characterized in that, described biological activity heterologous polypeptide forms disulfide linkage in described bacterial cell tenuigenin.

9. the method for claim 1, it is characterized in that, second kind of heterologous polypeptide expressed in described bacterial cytoplasm and linked to each other with described biological activity heterologous polypeptide in described tenuigenin, wherein said second kind of heterologous polypeptide lacks described leading peptide, and wherein said leading peptide instructs described second kind of heterologous polypeptide by exporting through the protein of double arginine transposition approach, and described second kind of heterologous polypeptide links to each other with described biological activity heterologous polypeptide.

10. the method for claim 1 is characterized in that, described bacterial cell is a Gram-negative bacteria.

11. the method for claim 1 is characterized in that, described bacterial cell is selected from intestinal bacteria trxB mutant strain, intestinal bacteria gor mutant strain and intestinal bacteria trxB gor double-mutant strain.

12. the method for claim 1 is characterized in that, two kinds of described biological activity heterologous polypeptides link to each other by at least one disulfide linkage.

Images