CN102533627A - Functional construction and application of SecB-mediated translated targeted path - Google Patents

Abstract

The invention relates to functional construction and application of a SecB-mediated translated targeted path, in particular to a method for constructing a chimeric SecA protein and co-expressing the chimeric SecA protein and a SecB protein in bacteria so as to increase the protein secretion efficiency of SecB gene deleted bacteria, a method for constructing the SecB-mediated translated targeted path in the SecB gene deleted bacteria as well as a method for enhancing the capability of the SecB gene deleted bacteria. In addition, the invention also relates to a related amino acid sequence and a nucleotide sequence, an expression vector and a system containing the amino acid sequence, the nucleotide sequence and/or the expression vector.

Description

The formation function and the application thereof of target approach after the translation of SecB mediation

Technical field

The invention belongs to gene engineering technology field, relate to chimeric SecA and based on chimeric SecA, by this technological method of structure of target approach after the translation of SecB mediation, and this technological method is produced the application in the secretory protein in industry.

Background technology

Bacterium is used for the albumen that large scale fermentation production has various using values widely, as: pharmaceutical protein, industrial enzymes etc.At present, be arranged in tenuigenin or substratum, can be divided into two types to production technology: a) with the mode productive target albumen of intracellular protein according to target protein after the fermentation ends; B) with the mode productive target albumen of extracellular protein (being secretory protein).Compare with the former; The latter is because target protein is arranged in substratum; Need not reclaim cell after the fermentation ends, need not take physics, chemical or biological method with lysis so that the release of target protein; Therefore simplify the separation and purification work of fermentation back target protein greatly, effectively controlled production cost [1].In addition, also has other advantage with the mode productive target albumen of extracellular protein, for example: can effectively reduce the proteic accumulation of tenuigenin internal object, thereby avoid the formation [2] of inclusion body; The albumen that allows to contain disulfide linkage can carry out correct folding etc. under oxidative environment.Therefore, the mode large scale fermentation productive target albumen with secretory protein is preferred option in industry.

Bacterium can be to be transported to the tenuigenin in the substratum of extracellular from its translation place with the albumen that contains signal peptide (being secretory protein), and this process is called protein secreting.In bacterium, the execution of this function mainly relies on Sec translocase (Sec translocase) and realizes, its core constitutes [3] by transmembrane protein passage SecYEG and molecular motor SecA (ATP enzyme), and is as shown in Figure 5.The new polypeptide chain that carries signal peptide need be transferred to from tenuigenin on the key ingredient SecA of Sec translocase, and this process is called target; Subsequently, provide at hydrolysising ATP under the situation of energy, SecA will treat that the excretory new polypeptide chain squeezes out tenuigenin via SecYEG albumen passage, thereby get into the cytolemma external space [4].Up to the present; As shown in Figure 5; The approach of the mediated targeted process of in bacterium, having identified has: 1) the common translation target approach of signal peptide identification particle (SRP) and acceptor (SR) mediation thereof, and the target of the secretory protein that responsible newborn signal peptide hydrophobicity is strong, i.e. SRP identification is exposed to the signal peptide on rrna surface; In translation,, the peptide chain in the translation is transferred to [5] on the Sec translocase through the interaction of SRP and its acceptor SR; 2) the common translation of SecA mediation/translation back target approach, the target of the secretory protein that responsible signal peptide hydrophobicity is general, promptly SecA need be by the newborn signal peptide [6] of other factor Direct Recognition; 3) target approach after the translation of SecB mediation; (promptly newborn secretion peptide chain is discerned by excretory system to assist those signal peptide efficient of SecA identification; In conjunction with and be transported to the efficient in the cytolemma outside; Influence factor comprises signal peptide characteristic and signal peptide downstream peptide section characteristic) target of low secretory protein; Thereby i.e. new life's secretion peptide chain combines to keep non-folding structure picture in translation latter stage or after finishing with SecB, the high-affinity of this binary complex and then dependence SecB and Sec translocase key ingredient SecA (by " electronegative zone " and SecA dimer C-terminal " positively charged zinc structural domain " i.e. interaction between " zinc-binding motif " mediation on SecB tetramer surface), thus give SecA with the peptide chain transfer; Accomplish target process [7,8].For specific secretory protein, rely on the characteristic which kind of target approach depends on its signal peptide, for example: signal peptide hydrophobicity [9,10].

The bacillus of Bacillus subtillis and relationship thereof is celebrated with its powerful PE ability, can the albumen direct secretion be made it to reach every liter the level [11,12] that restrains in substratum.This characteristic is produced highly beneficial for industrial enzymes; Therefore this bacterioid is widely used in industry, producing related prods [13; 14], for example: in existing commercialization enzyme, nearly 60% is to be produced by the bacillus fermentation that belongs to gram positive bacterium; Wherein the overwhelming majority is natural excretory oneself protein, as: glycase and proteolytic enzyme [2].Though be no lack of examples of many successful with Bacillus subtillis production external source secretory protein; Yet compare with endogenous secretory protein; In most cases the efficient of Bacillus subtillis secretion foreign protein is still low, and the albumen in those eukaryotes sources especially is on this layer meaning; This has limited its widespread use [15,16] in industry.Research shows that the factor of restriction foreign protein secernment efficiency mainly is target efficient and proteasome degradation, Given this; The researchist has been developed various strategies to strengthen the secretion capacity of Bacillus subtillis to foreign protein; For example: optimize signal peptide, cross the expression target factor (SRP), cross expression Sec translocase component; Cross the bacterial strain etc. of expressing mate molecule and using the proteolytic enzyme defective, and these strategies have also been applied for corresponding patent [1].

Along with the research of bacterioprotein secretory mechanism on molecular level deepens continuously, people are also deepening constantly its understanding.Bacterium is in order to adapt to specific living environment better; Protein secreting system (being called for short the Sec system) based on the Sec translocase demonstrates certain difference between different species in the process of evolving, for example: the difference [18] of difference of signal peptide characteristic [17] and Sec system etc.The existence of these differences has caused the external source secretory protein in heterologous host, efficiently to be secreted as endogenous secretory protein just.If can overcome these differences, be expected to fundamentally solve the low problem of foreign protein secernment efficiency in heterologous host.The result who has delivered shows, compares with intestinal bacteria, and Bacillus subtillis has lacked target approach [18] after the translation of SecB mediation.Yet because its signal peptide has more positive charge [17] than intestinal bacteria signal peptide hydrophobicity is stronger with the N end, so endogenous secretory protein can effectively be discerned [19] by SRP.Consider the signal peptide significant difference [17] of gram negative bacterium and Eukaryotic signal peptide and gram positive bacterium; We infer that these external source secretory proteins can't effectively be discerned by SRP or SecA in Bacillus subtillis; Cause the secretory protein precursor in tenuigenin, to accumulate and/or cause the protein quality system it is degraded; The target inefficiency that causes these foreign proteins finally causes secernment efficiency low [20,21].Produce foreign protein in order to impel Bacillus subtillis in industry, to be widely used in secretion, must solve the problem of target inefficiency.A way is to transform signal peptide or screening Optimal Signals peptide [22-24], thereby obtains being fit to the signal peptide of target protein.Because specific target protein needs the specific signal peptide just can reach ideal target efficient, a shortcoming of this strategy is exactly to different purpose albumen, need do the specific signal peptide and transform or screen.Being based on the molecular level the understanding of protein secreting, considering 1) the target approach can be assisted the secretory protein of SecA recognition signal peptide inefficiency after the translation of SecB mediation; 2) inefficiency of the signal peptide of external source secretory protein (as deriving from gram negative bacterium and eukaryote) in Bacillus subtillis; So the present invention attempts target approach after the translation of in Bacillus subtillis reconstruct SecB mediation; In the hope of strengthening the secretion capacity of this bacterium to the external source secretory protein; The maltose binding protein two mutants (Ma1E11) of promptly originating with intestinal bacteria; The fusion rotein (MalE-PhoA) of SEAP (PhoA) and wild-type maltose binding protein and SEAP is as the external source secretory protein; Studied beSecA and ecSecB can be in Bacillus subtillis after the translation of reconstruct SecB mediation the existence of target approach and this target approach can strengthen the secretion capacity of host to the external source secretory protein, accomplished the present invention thus.

Summary of the invention

The present invention provides a kind of method that increases bacterium secretory protein efficient, and this method comprises:

Chimeric SecA albumen of coexpression and SecB albumen in this bacterium, thus in this host, make up target approach after the translation of SecB mediation, increase the efficient of this bacterium secretory protein thus;

Wherein, the sequence allos of said chimeric SecA proteic " zinc-binding motif " and this proteic other part, and said chimeric SecA albumen can combine said SecB albumen.

The present invention provides the method for target approach after a kind of translation that in bacterium, makes up the SecB mediation, and this method comprises:

Chimeric SecA albumen of coexpression and SecB albumen in this bacterium, thereby target approach after the translation that structure SecB mediates in this host;

Wherein, the sequence allos of said chimeric SecA proteic " zinc-binding motif " and this proteic other part, and said chimeric SecA albumen can combine said SecB albumen.

The present invention provides a kind of method that improves the bacterioprotein secretion capacity, and this method comprises:

Chimeric SecA albumen of coexpression and SecB albumen in this bacterium, thus target approach after the translation that structure SecB mediates in this host improves this protein of bacteria secretion capacity thus;

Wherein, the sequence allos of said chimeric SecA proteic " zinc-binding motif " and this proteic other part, and said chimeric SecA albumen can combine said SecB albumen.

In one embodiment, said albumen is selected from natural secretory protein or artificial secretory protein, and wherein natural secretory protein is preferably the natural secretory protein of external source.

In one embodiment, said albumen is selected from the natural secretory protein of external source, comprises lytic enzyme (for example: proteolytic enzyme, glycase or lypase), antibody, Interferon, rabbit and growth factor.

In one embodiment, said albumen is fusion rotein, is preferably with maltose binding protein to merge and the fusion rotein of formation.

In one embodiment, said bacterium is the bacterium of natural disappearance secB gene, and the natural secA gene that exists of said bacterium.

In one embodiment, said bacterium is for being selected from the bacterium of Bacillus (Bacillus), Corynebacterium (Corynebacterium), mycobacterium (Mycobacterium), streptomyces (Streptomyces), Staphylococcus (Stapbylococcus), lactobacillus (Lactobacillus), streptococcus (Streptococcus) or fusobacterium (Clostridium).

In one embodiment, said bacterium is selected from subtilis (Bacillus subtilis), bacillus licheniformis (Bacillus licheniformis), Bacillus megatherium (Bacillus megaterium), bacillus brevis (Bacillus brevis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus pumilus (Bacillus pumilus) or Tribactur (Bacillus thuringiensis).

In one embodiment, said in this bacterium the chimeric SecA albumen of coexpression and SecB albumen comprise:

Structure contains chimeric secA expression carrier and contains the secB expression carrier; Wherein, Said chimeric secA gene is that artificial reconstructed said bacterium secA gene gets; The encoding sequence that is said bacterium secA gene " zinc-binding motif " is replaced by the encoding sequence of external source secA gene " zinc-binding motif ", thereby possesses the proteic ability of the said SecB of combination;

Contain chimeric secA expression carrier and contain the secB expression carrier and transform this bacterium with said, thus in this bacterium chimeric SecA albumen of coexpression and SecB albumen.

In one embodiment, said secB gene comes from the bacterium with said bacterium different genera.

In one embodiment, said secB gene and the encoding sequence of " zinc-binding motif " of said secA gene derive from the bacterium of identical kind.

In one embodiment, described external source secA coded by said gene SecA proteic " zinc-binding motif " is terminal last 18～60 amino acid of external source SecA protein carboxyl groups.

In one embodiment, said external source secA gene is intestinal bacteria secA gene or hemophilus influenzae secA gene.

In one embodiment, said external source secB gene is intestinal bacteria secB gene or hemophilus influenzae secB gene.

The present invention provides a kind of aminoacid sequence, and this aminoacid sequence contains SEQ ID NO:29 or the described aminoacid sequence of SEQ ID NO:31.

The present invention provides a kind of nucleotide sequence, said nucleotide sequence coded power aminoacid sequence of the present invention.

The present invention provides a kind of construction, and said construction contains nucleotide sequence of the present invention.

The present invention provides a kind of system, and said system contains:

(a) chimeric secA gene or contain the construction of this chimeric secA gene; Or the chimeric SecA albumen of said chimeric secA coded by said gene; Wherein, Other part allos of the encoding sequence of " zinc-binding motif " of said chimeric secA gene and this chimeric secA gene, chimeric SecA albumen possess the proteic ability of the said SecB of combination; With

(b) secB gene or contain the construction of this chimeric secB gene, or the SecB albumen of said secB coded by said gene possess the protein bound characteristic by said chimeric SecA.

Description of drawings

Fig. 1: pSJ3-ecSecA plasmid synoptic diagram.

Fig. 2: pMA5-ecMalE11 plasmid synoptic diagram.

Fig. 3: pAX01-ecSecB plasmid synoptic diagram.

Fig. 4: pOE-beSecA plasmid synoptic diagram.

Fig. 5: Bacillus subtillis protein target approach synoptic diagram.Semi-circular dash area be the present invention relates to based on chimeric SecA, by target approach after the translation of SecB mediation.

Fig. 6: the terminal decision of SecA protein carboxyl groups SecA-SecB specificity interacts.

Fig. 7: the chimeric SecA albumen of coexpression (beSecA) can strengthen the ability that Bacillus subtillis secretes foreign protein MalE11 with SecB albumen (ecSecB).

Target approach after the translation of the efficient secretion dependence SecB mediation of Fig. 8: MalE11.

The target approach can strengthen the ability of Bacillus subtillis secretion foreign protein PhoA and MalE-PhoA fusion rotein after the translation of Fig. 9: SecB mediation.

Figure 10: terminal " zinc-binding motif " decision SecA-SecB specificity of SecA protein carboxyl groups interacts.

Embodiment

In this application, said " host " or " protein production bacterium " or " bacterium " comprise range protein production bacterium, comprise gram positive bacterium and gram negative bacterium.Preferably, " host " of the present invention or " protein production bacterium " or " bacterium " refer to mainly that its wild-type contains SecA albumen but the proteic bacterium of disappearance SecB.More preferably; " host " of the present invention or " protein production bacterium " or " bacterium " mainly are gram positive bacteriums; Comprise that those come from the bacterium of Bacillus (Bacillus), Corynebacterium (Corynebacterium), mycobacterium (Mycobacterium), streptomyces (Streptomyces), Staphylococcus (Staphylococcus), lactobacillus (Lactobacillus), streptococcus (Streptococcus), fusobacterium (Clostridium) or other genus, be specially the bacterium of subtilis (Bacillus subtilis), bacillus licheniformis (Bacillus licbeniformis), Bacillus megatherium (Bacillus megaterium), bacillus brevis (Bacillus brevis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus pumilus (Bacillus pumilus), Tribactur (Bacillus thuringiensis) or other kinds.

Among the application, " external source " is for endogenous, refers to that certain component derives from the bacterium of said host of non-the application or protein production bacterium, mainly refers to the bacterium from different genera.

Adopt the application's method, can transform above-mentioned bacterium, especially the ability and the efficient of external source secretory protein are improved to make the production secretory protein.Can adopt the albumen of the application's method production to include but not limited to natural secretory protein (the natural signal peptide that contains); Be the natural excretory albumen of host bacterium or protein production bacterium self (being endogenous secretory protein); Natural secretory protein (being the external source secretory protein) with source of species outside this host bacterium or the protein production bacterium; The signal peptide that these albumen carry can be a wild-type, also can be the signal peptide that is obtained behind artificial replacement or the sudden change wild-type signal peptide.

Except that these natural secretory proteins; Can adopt the albumen of the application's method production also can be artificial constructed secretory protein, and promptly natural non-secretory protein (natural no signal peptide) is utilizing artificial " the artificial secretory protein " that obtains behind the signal peptide that add of genetic engineering technique.Non-secretory protein both can be the albumen of the natural production of host bacterium self, also the albumen produced of the species outside the host bacterium.

In addition, can adopt the albumen of the application's method production can be fusion rotein.For example, said endogenous secretory protein, external source secretory protein and artificial secretory protein can be expressed in host bacterium of the present invention or protein production bacterium and secrete with the form of various fusion roteins.Can contain the expression vector of the encoding sequence of the fusion rotein that this endogenous secretory protein, external source secretory protein or artificial secretory protein and other albumen forms, this carrier is changed over to express in host bacterium or the protein production bacterium and secrete then through structure.In a preferred embodiment, the fusion rotein of preferred above-mentioned endogenous or external source secretory protein, natural or artificial secretory protein and maltose binding protein formation.The method that makes up and transform all is that this area is conventional.

In a preferred embodiment, can adopt method of the present invention or bacterium excretory albumen to comprise various industrial enzymes (for example: proteolytic enzyme, glycase, lypase etc.) and various pharmaceutical albumen (for example antibody, Interferon, rabbit, growth factor etc.) [seeing document 2,25,26].These industrial enzymes or pharmaceutical albumen can fusion rotein form (for example, merging) with maltose binding protein by method of the present invention or bacterium secretion.

The method that the present invention transforms bacterium includes but not limited to: structure contains chimeric secA expression carrier and contains the SecB expression carrier; Contain chimeric secA expression carrier and contain the secB expression carrier and transform this bacterium with said then, thus in this bacterium chimeric SecA albumen of coexpression and SecB albumen.

Among the present invention; Chimeric secA gene is that artificial reconstructed host bacterium secA gene gets; The encoding sequence that is said bacterium secA gene " zinc-binding motif " is replaced by the encoding sequence of external source secA gene " zinc-binding motif ", thereby possesses the proteic ability of the said SecB of combination.

Among this paper; SecA albumen " zinc-binding motif " refers to CXCX8C (C/X) that SecA protein carboxyl groups (C) is terminal and the conservative amino acid residues that closes on thereof; Be responsible for mediation and the proteic interaction of SecB, should " zinc-binding motif " generally be arranged in terminal last about 40 amino acid residue sequences of SecA protein carboxyl groups.Can use SecA proteic " zinc-binding motif " replacement host or the protein production bacterium SecA proteic " zinc-binding motif " of the bacterium of the target approach that comes self-contained SecB mediation.As long as the zone that is replaced comprises " zinc-binding motif ", the zone of replacement can be " zinc-binding motif ", also can be longer zone, like terminal last about 60,55,50,45 amino acid or shorter of SecA PROTEIN C.Therefore, in certain embodiments, with terminal last 18～60 amino acid of external source SecA PROTEIN C (for example, last 18～40,20～35,22～35,22～32 amino acid) replacement host or the terminal corresponding section of protein production bacterium SecA PROTEIN C.In other embodiments, the sequence that is used to replace is not necessarily started at from last terminal amino acid of this external source SecA PROTEIN C.For example; The sequence that is used to replace can be the arbitrary amino acid fragment in terminal the 2nd～40 amino acids of this external source SecA PROTEIN C, the 2nd～35 amino acids, the 2nd～32 amino acids, the 3rd～40 amino acids, the 3rd～35 amino acids etc. and these scopes, as long as sequence or the amino acid fragment replaced still keep the biological function of " zinc-binding motif ".The chimeric SecA that this method makes up possesses combination and the proteic ability of its " zinc-binding motif " homologous SecB.For example, for example can use shown in Fig. 6 A from bacterium SecA proteic " zinc-binding motif " such as intestinal bacteria, hemophilus influenzae, A.Tumefaciens, P.fluorescens, R.etli, A.Pleuropneumoniae replacement host or protein production bacterium SecA proteic " zinc-binding motif ".In a specific embodiment, the sequence from bacteriums such as intestinal bacteria, hemophilus influenzae, A.Tumefaciens, P.fluorescens, R.etli, A.Pleuropneumoniae that can use Fig. 6 A specifically to list is replaced SecA proteic " zinc-binding motif " in the host bacterium.

Can adopt the conventional method in this area to carry out above-mentioned replacement as merging PCR.For example, the said structure of the application embodiment part carries the chimeric secA gene of external source " zinc-binding motif " encoding sequence, and then in host or protein production bacterium, expresses this chimeric SecA albumen.

Can adopt known material in this area (Bacillus subtillis expression vector) and technology (PCR and molecular cloning) to make up the present invention contains chimeric secA expression carrier and contains external source secB expression carrier.Adopt subsequently and well known in the artly affiliated expression vector is incorporated in the Bacillus subtillis cell such as chemical conversion process.Be applicable to that expression vector of the present invention can be integrated expression vector and rf expression vector, the former is like pAX01, pA-spac or pDG1661 etc.; The latter such as pUB110 series derivatives plasmid (pMA5 or pWB980) or pBS72 series derivatives plasmid (pHCMC05 or pOE) etc.In addition, the visible reference [14] of a large amount of operable expression vectors.

Whether the bacterium that can adopt this area ordinary method to detect to be transformed said chimeric SecA albumen of stably express and external source SecB albumen.These methods comprise the SDS-PAGE and the immune marking subsequently, and are of embodiment.

Among this paper; SecB gene or its proteins encoded preferably derive from the encoding sequence that makes up chimeric secA gene used " zinc-binding motif " but are not limited to same species or nearly source species, and the standard of selection is that the SecB that is selected for use must carry out functional interaction in vivo with chimeric SecA.

In a preferred implementation, preferably be used for making up the proteic external source of chimeric SecA " zinc-binding motif " and this external source SecB homology, promptly from identical bacterium, as all from intestinal bacteria or hemophilus influenzae.Certainly, from " zinc-binding motif " of different bacterium as long as and SecB can guarantee that chimeric SecA and SecB can carry out functional interaction in vivo, equally also can in same host or protein production bacterium, work, improve PE efficient.

Among this paper, SEQ ID NO:28 has shown the nucleotide sequence of chimeric secA gene, and wherein the 2428th～2526 is the corresponding section of intestinal bacteria secA gene.SEQ ID NO:29 has shown the aminoacid sequence of chimeric SecA.SEQ ID NO:30 has shown the nucleotide sequence of another chimeric secA gene of the present invention, the wherein chimeric corresponding section of intestinal bacteria secA gene, and SEQ ID NO:31 shows its aminoacid sequence.

Therefore, the application also comprises a kind of aminoacid sequence, and this sequence contains the aminoacid sequence shown in SEQ ID NO:29 or the SEQID NO:31.The application also comprises a kind of nucleotide sequence, and this sequence encoding the application contains the aminoacid sequence of the sequence shown in SEQ ID NO:29 or the SEQ ID NO:31.The application also comprises the construction that contains the said nucleotide sequence of the application.In a preferred embodiment, said construction can be a kind of carrier.In preferred embodiment, said construction can be a kind of expression vector, is used for the chimeric SecA albumen in host or protein production bacterium expression the application.Can adopt the conventional technique means in this area to make up the expression vector that contains said Nucleotide.

The application also comprises the purposes of above-mentioned aminoacid sequence, nucleotide sequence and construction; For example, be used to increase protein production bacterium secretion foreign protein efficient, be used to improve the production of external source secretory protein and be used to make up compare protein production bacterium that its foreign protein throughput is improved etc. with its wild-type contrast.

The application also comprises a kind of system, and this system contains chimeric secA gene as herein described and secB gene, and/or chimeric SecA albumen and SecB albumen.In a specific embodiment; Said system is a kind of cell or bacterium; Telescoping part in the said chimeric secA gene (for example: the encoding sequence of " zinc-binding motif "); With said secB gene with respect to all being that telescoping part and SecB albumen in external source and the said chimeric SecA albumen also all is external source for this cell or bacterium for this cell or the bacterium.

In a specific embodiment; Said system contains the nucleotide sequence of SEQ ID NO:28 or 30 described nucleotide sequences and the said intestinal bacteria secB of GenBank:M24489.1 gene, the perhaps coded aminoacid sequence of aminoacid sequence shown in the SEQ ID NO:29 or 31 and GenBank:M24489.1.In a specific embodiment, said system is a Bacillus subtillis.In other embodiments, said system is bacillus licheniformis (Bacillus licheniformis), Bacillus megatherium (Bacillus megaterium), bacillus brevis (Bacillus brevis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus pumilus (Bacillus pumilus) or Tribactur (Bacillus thuringiensis) etc.

Below will carry out detailed description to the present invention with the mode of specific embodiment.Should be understood that these embodiment only are illustrative.In addition, among this paper, terms such as " containing ", " comprising " also comprised in this article " by ... form ", " by ... constitute " etc. implication.The experimental technique of unreceipted actual conditions in the following example carries out according to routine operation usually, for example with reference to " molecular cloning: the laboratory manual " third edition of press of cold spring harbor laboratory publication or the condition of being advised according to used article manufacturer.

One. materials and methods

1) bacterial strain and plasmid

Plasmid pSJ2, pSJ3 and pSJ4 (document that sees reference [27]) are pET21a (Novagen) plasmid of deriving, and are used at recombinant expressed SecA of intestinal bacteria and SecB albumen.

Plasmid pMA5 provides [28] by Dartois, and kalamycin resistance is used for expressing the external source secretory protein Bacillus subtillis.

Plasmid pAX01 is provided by Bacillus subtilus DSMZ (BGSC), and the Oxacyclotetradecane,erythromycin deriv resistance is used for expressing intestinal bacteria SecB albumen Bacillus subtillis.

Plasmid pOE is a basic framework with pMD18 (TaKaRa), introduces the Bacillus subtillis that comes from the pHCMC05 [29] and duplicates unit and chlorampenicol resistant mark; Introduce simultaneously the expression casette that HpaII promotor and colibacillary trpA terminator sequence on the pMA5 are formed again.This plasmid is used for expressing SecA albumen Bacillus subtillis.

Cloning host bacillus coli DH 5 alpha and protein expression host e. coli BL21 (DE3) are provided by Novagen.

Bacillus subtillis 168 (Bacillus subtilis 168) is provided by Bacillus subtilus DSMZ (BGSC).

2) plasmid construction

A) pSJ3-ecSecA, pSJ3-bsSecA, pSJ3-bhSecA and pSJ3-beSecA

Plasmid pSJ3-ecSecA coding intestinal bacteria SecA albumen; With the bacillus coli gene group is template, and primer is to P1 (SEQ ID NO:1) and P2 (SEQ ID NO:2) amplification secA gene, and pSJ3 packs into behind NdeI and the BamHI double digestion; Obtain pSJ3-ecSecA, its plasmid synoptic diagram is as shown in Figure 1.Plasmid pSJ3-bsSecA coding Bacillus subtillis SecA albumen; With the Bacillus subtillis genome is template; Primer is to P3 (SEQ ID NO:3) and P4 (SEQ ID NO:4) amplification secA gene, and the pSJ3 that packs into behind BamHI and the XhoI double digestion obtains pSJ3-bsSecA.

Plasmid pSJ3-bhSecA coding (Bacillus subtillis-hemophilus influenzae) chimeric SecA albumen is bhSecA, this albumen by the 1st in Bacillus subtillis SecA albumen to the 867th in the 809th amino acids and hemophilus influenzae SecA albumen to the fusion of the 901st amino acids and get.This plasmid adopts the large primer PCR technique construction; Process is following: with the hemophilus influenzae genome is template, and primer is to the 867th encoding sequence to the 901st amino acids of P5 (SEQ ID NO:5) and P6 (SEQ ID NO:6) amplification hemophilus influenzae SecA albumen.Reclaiming this segment as big primer, with the P3 collocation, is that template amplification obtains the proteic encoding sequence of bhSecA with plasmid pSJ3-bsSecA, and the pSJ3 that packs into behind BamHI and the HindIII double digestion obtains pSJ3-bhSecA.In like manner; Plasmid pSJ3-beSecA coding (Bacillus subtillis-intestinal bacteria) chimeric SecA is a beSecA albumen, this albumen by the 1st in Bacillus subtillis SecA albumen to the 870th in the 809th amino acids and intestinal bacteria SecA albumen to the fusion of the 901st amino acids and get.With the bacillus coli gene group is template, and primer is to the 870th encoding sequence to the 901st amino acids of P7 (SEQ ID NO:7) and P2 amplification intestinal bacteria SecA albumen.Reclaiming this segment as big primer, with the P3 collocation, is that template amplification obtains the proteic encoding sequence of beSecA with plasmid pSJ3-bsSecA, and the pSJ3 that packs into after the BamHI enzyme is cut obtains pSJ3-beSecA.

B) pSJ4-hiSecB and pSJ2-ecSecB

The structure of plasmid pSJ4-hiSecB is seen document [30], this plasmid-encoded hemophilus influenzae SecB albumen.Plasmid pSJ2-ecSecB coding intestinal bacteria SecB albumen is template with the bacillus coli gene group, and primer is to P8 (SEQ ID NO:8) and P9 (SEQ ID NO:9) amplification secB gene, and BamHI and the HindIII couple of pSJ2 that packs into after cutting obtains pSJ2-ecSecB.

Above a) and b) in the plasmid that makes up be used in intestinal bacteria purifying corresponding proteins, these purified proteins are used for external combination experiment subsequently and test with isothermal titration.

C) pMA5-ecMalE11, pMA5-ecPhoA and pMA5-(ecMalE-PhoA)

The two mutants of plasmid pMA5-ecMalE11 coding intestinal bacteria maltose binding proteins (MalE), i.e. MalE11 albumen, the N-terminal of this two mutants carries 3 amino acid whose replacements (shown in Fig. 7 A).Sudden change is introduced by overlap extension pcr; The plasmid construction process is following: with the bacillus coli gene group is template; Primer is to P10 (SEQ ID NO:10) and P11 (SEQ ID NO:11) amplification malE gene signal peptide coding region, and primer is to P12 (SEQ ID NO:12) and P13 (SEQ ID NO:13) amplification malE gene mature peptide section coding region.With above-mentioned two purified PCR mixture of products is template; Primer obtains the proteic encoding sequence of total length MalE11 (sudden change is introduced by P10 and P12 respectively) to P10 and P13 amplification; PMA5 packs into behind NdeI and the HindIII double digestion; Obtain pMA5-ecMalE11, its plasmid synoptic diagram is as shown in Figure 2.

The plasmid pMA5-ecPhoA escherichia coli alkaline phosphatase (PhoA) of encoding; Its building process is following: with the bacillus coli gene group is template; Primer is to P14 (SEQ ID NO:14) and P15 (SEQ ID NO:15) amplification phoA gene; The pMA5 that packs into behind NdeI and the HindIII double digestion obtains pMA5-ecPhoA.

The maltose binding protein in plasmid pMA5-(ecMalE-PhoA) coding intestinal bacteria source and the fusion rotein (MalE-PhoA) of SEAP, the encoding sequence of this fusion rotein adopts overlap extension pcr to make up.The plasmid construction process is following: with the bacillus coli gene group is template, and primer does not contain the malE gene of terminator codon to P16 (SEQ ID NO:16) and P17 (SEQ ID NO:17) amplification; Primer is to the encoding sequence of P18 (SEQ ID NO:18) with P15 amplification coding PhoA mature peptide section zone.With above-mentioned two purified PCR mixture of products is template, and primer obtains the proteic encoding sequence of total length MalE-PhoA to P16 and P15 amplification, and the pMA5 that packs into behind NdeI and the HindIII double digestion obtains pMA5-(ecMalE-PhoA).

D) pAX01-ecSecB, pAX01-ecSecBL75Q, pAX01-ecSecBE77K and pAX01-ecSecBL75Q&E77K

Plasmid pAX01-ecSecB coding intestinal bacteria SecB albumen; With the bacillus coli gene group is template, and primer is to P19 (SEQ ID NO:19) and P20 (SEQ ID NO:20) amplification secB gene, and pAX01 packs into after the BamHI enzyme is cut; Obtain pAX01-ecSecB, its plasmid synoptic diagram is as shown in Figure 3.

The proteic two mutants SecBL75Q of plasmid pAX01-ecSecBL75Q coding intestinal bacteria SecB; Sudden change is introduced by overlap extension pcr; The plasmid construction process is following: with the bacillus coli gene group is template; Primer is to the segment at the P19 and P21 (SEQ ID NO:21) the amplification secB gene mutation site upper reaches, and primer is to the segment of P22 (SEQ ID NO:22) with P20 amplification secB gene mutation site downstream, and sudden change is introduced by P22.With above-mentioned two purified PCR mixture of products is template, and primer obtains the proteic encoding sequence of total length SecBL75Q to P19 and P20 amplification, and the pAX01 that packs into after the BamHI enzyme is cut obtains pAX01-ecSecBL75Q.With same method; P23 (SEQ ID NO:23) and P24 (SEQ ID NO:24) are respectively applied for the structure of pAX01-ecSecBE77K and pAX01-ecSecBL75Q&E77K, these two plasmids encode respectively proteic two mutants SecBE77K of intestinal bacteria SecB and SecBL75Q/E77K.

E) pOE-beSecA and pOE-bsSecA.

Plasmid pOE-beSecA coding (Bacillus subtillis-intestinal bacteria) chimeric SecA is a beSecA albumen; With plasmid pSJ3-beSecA is template; Primer obtains the proteic encoding sequence of beSecA to P25 (SEQ ID NO:25) with P26 (SEQ ID NO:26) amplification; The pOE that packs into behind KpnI and the SacII double digestion obtains pOE-beSecA, and its plasmid synoptic diagram is as shown in Figure 4.

Plasmid pOE-bsSecA coding Bacillus subtillis SecA albumen is template with the Bacillus subtillis genome, and primer is to P25 and P27 (SEQ ID NO:27) amplification secA gene, and the pOE that packs into behind KpnI and the SacII double digestion obtains pOE-bsSecA.

3) culture condition

As do not have specified otherwise, and all adopt the LB substratum, add suitable microbiotic, 37 degrees centigrade of shaking culture are spent the night.Antibiotic concentration is: penbritin 100ug/ml, kantlex 100ug/ml, paraxin 5ug/ml, Oxacyclotetradecane,erythromycin deriv 5ug/ml.

4) method for transformation

Intestinal bacteria transform adopts sophisticated calcium method to transform, and sees " molecular cloning " third edition.Bacillus subtillis adopts widely used inorganic salt nature competence method to carry out the conversion [31] of plasmid.

5) SDS-PAGE, laboratory reference " molecular cloning " third editions such as Western Blotting.

6) SEAP enzyme activity determination reference [32].

7) proteic purifying of SecA albumen and SecB and external combination experiment etc. are with reference to [27]

Two. result and discussion

Embodiment one

The terminal decision of SecA protein carboxyl groups SecA-SecB specificity interacts.In the bacterium of disappearance secB gene,, lost the proteic ability of combination SecB gradually along with terminal " zinc-binding motif " the continuous accumulation of " detrimental mutation " during evolution of SecA protein carboxyl groups.With this type SecA protein carboxyl groups terminal " zinc-binding motif " replace with can with the proteic corresponding section of the SecA of SecB protein-interacting; Can make to combine the proteic SecA albumen of SecB to obtain to combine the proteic ability of SecB originally, be that example is explained with Bacillus subtillis SecA below.

Fig. 6 A shows that the sequence of the SecA albumen " zinc-binding motif " of different biogenetic derivations compares.This shows that during evolution, though this motif demonstrates high conservative property, sudden change also is significant.For the bacterium of those disappearances secB gene, like subtilis, sudden change possibly cause its SecA proteins lose to combine the proteic ability of SecB.

Fig. 6 B has shown hemophilus influenzae SecA (Haemophilus influenzae SecA; Be abbreviated as hiSecA); Intestinal bacteria SecA (Escherichia coli SecA; Be abbreviated as ecSecA); Bacillus subtillis SecA (Bacillus subtilis SecA is abbreviated as bsSecA) and two C-terminal aminoacid sequences that chimeric SecA is the chimeric SecA of withered grass brood cell bacillus-hemophilus influenzae (being abbreviated as bhSecA) and Bacillus subtillis-intestinal bacteria SecA (being abbreviated as beSecA).Chimeric position takes place in arrow indication part when representing to make up chimeric SecA.

Fig. 6 C shows different SecA and the external situation that combines of SecB, and the result shows that bsSecA can not combine hemophilus influenzae SecB (hiSecB), can not combine intestinal bacteria SecB (ecSecB), sees swimming lane 1 and 2.This and Bacillus subtillis lack the true consistent of secB gene, and the proteic ability of combination SecB has been lost in terminal " zinc-binding motif " the continuous accumulation of " detrimental mutation " during evolution of its SecA protein carboxyl groups gradually.Yet; The chimeric SecA that bsSecA proteic " zinc-binding motif " is replaced with the corresponding section acquisition of hiSecA or ecSecA is that bhSecA and beSecA have just obtained to combine and the proteic ability of its " zinc-binding motif " homologous SecB at least; See swimming lane 3,4 and 6.This result clearly illustrates that the terminal decision of SecA protein carboxyl groups SecA-SecB specificity interacts, and can change the characteristic that it combines SecB through displacement SecA protein carboxyl groups terminal " zinc-binding motif ".

Explain that in order further to produce evidence chimeric SecA can effectively combine SecB albumen, we have measured different SecA and the dissociation constant between the ecSecB.Fig. 6 D shows that the dissociation constant between the beSecA/ecSecB and suitable over against the dissociation constant of shining between the ecSecA/ecSecB is far below the dissociation constant between the bsSecA/ecSecB.

Embodiment two

Can interact with external source SecB based on chimeric SecA; In theory in the bacterium of secB genetically deficient chimeric SecA albumen of coexpression and external source SecB albumen (both can interact) can reconstruct SecB the target approach of mediation, can strengthen the efficient that the host secretes foreign protein.Be that example is explained with coexpression beSecA and ecSecB in Bacillus subtillis below, the external source secretory protein of selecting for use is MalE11.

MalE11 is the two mutants of intestinal bacteria source MalE; The mature peptide section zone that the signal peptide n-quadrant of this two mutants only has 1 clean positive charge (wild-type is 3 clean positive charges) and is right after signal peptide also has 1 clean positive charge (the wild-type net charge is 0); So called after MalE11; Shown in Fig. 7 A; The secernment efficiency of this two mutants in Bacillus subtillis compared with wild-type and decreased, so target approach functional after adopting this two mutants with the translation of the outstanding SecB of demonstration mediation among the present invention promptly strengthens the efficient that the host secretes foreign protein.

BsSecA or beSecA or empty carrier pOE will encode; Coding ecSecB or empty carrier pAX01 are transformed in the Bacillus subtillis with three plasmids of pMA5 of coding MalE11 simultaneously; Obtain 6 strains and express different SecA respectively, the bacterial strain of ecSecB and MalE11 combination.These strain bacterium are cultivated 15 hours in the LB substratum that interpolation 0.5% wood sugar induces ecSecB to express after, sampling analysis, the result is shown in Fig. 7 B, and single expression ecSecB, bsSecA or beSecA can't increase the efficient (the 2nd, 3 and 5 group) that the host secretes MalE11.Under the situation of coexpression bsSecA and ecSecB (the 4th group), the secernment efficiency of MalE11 has a little raising, but the amplitude that improves is weaker than the situation (the 6th group) of coexpression beSecA and ecSecB far away.Under the situation of coexpression beSecA and ecSecB, the efficient that the host secretes MalE11 increases considerably, and the increase of MalE11 secretory volume is more than at least 1 times.We can see and accumulated a large amount of MalE11 maturation proteins in the substratum, and only accumulate a small amount of MalE11 precursor protein in the tenuigenin.These results clearly illustrate that the coexpression of beSecA and ecSecB can be in Bacillus subtillis target approach after the translation of reconstruct SecB mediation, the existence of this approach can increase the efficient that the host secretes foreign protein.

Embodiment three

The invention provides two " reverse side " evidences; Further support conclusion of the present invention; Be the coexpression of beSecA and ecSecB can be in Bacillus subtillis target approach after the translation of reconstruct SecB mediation, the existence of this approach can increase the efficient that the host secretes foreign protein.

The expression of ecSecB is controlled by wood sugar inductive promotor Pxy1; We regulate and control the expression level of ecSecB through the concentration of adjustment inductor wood sugar in the experiment of this group; Be that xylose concentration is followed successively by 0.5%; 0.1%, 0.05% and 0% (Fig. 8 A is from the 3rd group to the 6th group), and then investigate of the influence of the expression level of ecSecB to the MalE11 secernment efficiency.Consistent with expection, Fig. 8 A clearly illustrates that along with the ecSecB expression level descends gradually, the host secretes the efficient of MalE11 and also successively decreases gradually, demonstrates linear relationship.

Have more the experiment that convictive evidence comes from the ecSecB two mutants.The point mutation L75Q of SecB and E77K can destroy specificity interaction between the SecA-SecB, but do not influence the activity that SecB combines newborn secretion peptide chain.Adopt these two mutants, whether the efficient secretion that can investigate MalE11 relies on specificity interaction between the SecA-SecB.The ecSecB that will encode, ecSecB L75Q, the pAX01 carrier of ecSecB E77K or ecSecB L75Q&E77K are transformed into respectively in the Bacillus subtillis of expressing beSecA and MalE11, incubated overnight under suitable condition, and sampling analysis, the result is shown in Fig. 8 B.Compare with wild-type SecB (the 3rd group), SecB two mutants (the 4th, 5 and 6 group) can not be supported the efficient secretion of MalE11, explains that the efficient secretion of MalE11 relies on specificity interaction between the SecA-SecB, and this result is in full accord with expection.

Embodiment four

The target approach can increase the efficient that the host secretes foreign protein and has certain versatility after the translation of SecB mediation, is that intestinal bacteria source SEAP (PhoA) and maltose binding protein-alkaline phosphatase enzyme fusion proteins (MalE-PhoA) are that example is explained with the external source secretory protein below.

Be similar to MalE11, Fig. 9 A has studied the secretory volume of PhoA under different SecA and the ecSecB combination background in great detail, and the PhoA secretory volume can just be reflected by the PhoA enzyme activity in the substratum.Under the situation of single expression ecSecB, the secretory volume of PhoA does not increase (No. 2 compare with No. 1).It should be noted that no matter be single expression bsSecA, or beSecA (No. 3 with No. 1 compare with No. 5), the PhoA secretory volume all increases about 30%, and this is consistent with bibliographical information, and promptly SecA separately also can the newborn secretion of mediated targeted approach identification peptide chain.It is worth noting that more under the situation of beSecA and ecSecB coexpression, the PhoA secretory volume is increasing the secretory volume that has further increased PhoA on the basis of 30% (expression by beSecA causes) again, final PhoA secretory volume cumulative rises more than 60%.As " reverse side " evidence; The L75Q sudden change of ecSecB is owing to destroyed specificity interaction between the SecA-SecB; So the coexpression of beSecA and ecSecBL75Q can not make the cumulative rises of PhoA secretory volume reach more than 60%, 30% of increase is derived from the single expression (No. 7) of beSecA.In brief, above-mentioned these results prove once more the coexpression of beSecA and ecSecB can be in Bacillus subtillis target approach after the translation of reconstruct SecB mediation, the existence of this approach can increase the efficient that the host secretes foreign protein.

Immunoblotting confirms that the coexpression of beSecA and ecSecB has increased the secretory volume of PhoA shown in Fig. 9 B.Although its " apparent secernment efficiency " is very high, promptly do not detect a large amount of PhoA precursor (swimming lane 2) in the cellular component.The contriver infers under the situation of coexpression beSecA and ecSecB; The target efficient of PhoA increases greatly; Avoided part PhoA precursor in tenuigenin, to be degraded, thereby under the situation that the target approach that SecB mediates exists, the secretory volume of PhoA significantly increase.

Fig. 9 C shows the influence of the coexpression of beSecA and ecSecB to the MalE-PhoA secernment efficiency.Immunoblotting confirms under the situation of beSecA and ecSecB coexpression; The secernment efficiency of MalE-PhoA also is greatly improved; The MalE-PhoA secretory volume significantly increases in the substratum, and correspondingly the accumulation of its precursor in tenuigenin significantly reduces ( swimming lane 3 and 4 contrasts 1 and 2).In addition, enzyme activity determination has confirmed the result of the immune marking in the substratum, and promptly the MalE-PhoA secretory volume improves more than 70%.

Embodiment five

(document delivered at present of its exact boundary is report not also except comprising " zinc-binding motif " for the proteic telescoping part of using among the embodiment one to embodiment four of chimeric beSecA (32 amino acid of least significant end); But known last 22 amino acid contain this " zinc-binding motif "; See document [33]) outside, also comprise several amino acid of its upper reaches.Telescoping part (length can less than 22 amino acid or greater than 32 amino acid) is as long as comprise mediation and SecB interactional " zinc-binding motif " in theory; Chimeric SecA just can combine and the proteic ability of its " zinc-binding motif " homologous SecB at least; The coexpression of this chimeric SecA and this SecB can the translation of reconstruct SecB mediation in the bacterium of secB disappearance after the target approach; Strengthening the efficient that the host secretes foreign protein, is that example is explained below with bsSecA-R3.

Figure 10 A shows that bsSecA-R3 compares with beSecA, only replaces with terminal last 22 amino acid of bsSecA protein carboxyl groups the corresponding section of ecSecA.Replacement institute calling sequence is shown in SEQ ID NO:31.

Under the situation of coexpression bsSecA-R3 and ecSecB; The secernment efficiency of MalE11 will be apparently higher than the situation (the 4th group with respect to the 1st group) of coexpression bsSecA and ecSecB, thus this explanation bsSecA-R3 and ecSecB can the translation of reconstruct SecB mediation in the host after the target approach enhancing host efficient of secreting foreign protein.

Three. sum up

The present invention relates to the structure and the application thereof of target approach after the translation of SecB mediation.Consider 1) Bacillus subtillis is widely used in production (endogenous) secretory protein in industry; 2) because the signal peptide characteristic demonstrates significant difference, signal peptide inefficiency in Bacillus subtillis of external source secretory protein (as deriving from gram negative bacterium and Eukaryotic secretory protein) between different biological groups; 3) the target approach can effectively assist the new life of SecA recognition signal peptide inefficiency to secrete peptide chain after the translation of SecB mediation; 4) Bacillus subtillis disappearance SecB albumen; So the present invention attempts target approach after the translation of in Bacillus subtillis reconstruct SecB mediation; In the hope of increasing the efficient of this bacterium secretion external source secretory protein, further expand this bacterium and in industry, produce the application in the secretory protein.Because Bacillus subtillis SecA albumen (bsSecA; UniProtKB:P28366) can not with intestinal bacteria SecB albumen (ecSecB; UniProtKB:P0AG86) carry out effective interaction; Based on the interactional architecture basics of SecA-SecB, the present invention has made up beSecA, and promptly terminal last 32 amino-acid residues of bsSecA protein carboxyl groups are replaced into the proteic corresponding section of ecSecA; This part comprises mediation SecA-SecB interactional " zinc-binding motif ", thereby obtains effectively to combine the ability of ecSecB.Immunoblotting and/or enzyme activity determination show; Coexpression beSecA and ecSecB albumen in Bacillus subtillis; Target approach after the translation of can reconstruct originally non-existent SecB mediation; It is bud carbohydrate-binding protein two mutants (MalE11) that the existence of this approach can effectively increase Bacillus subtillis secretion foreign protein; The efficient of the fusion rotein (MalE-PhoA) of SEAP (PhoA) and maltose binding protein and SEAP, these proteic secretory volumes increase respectively more than 100%, 60% and 70% in the substratum.In addition, the functional of target approach after the translation of SecB mediation further verified in the control experiment that laterally arranges.These results clearly show, based on chimeric SecA, i.e. beSecA albumen, we in Bacillus subtillis successful reconstruct target approach after the translation of SecB mediation, this approach can increase the efficient that the host secretes foreign protein.In addition; Technological method provided by the invention also can be used for the bacterium of target approach after translation of other disappearance SecB mediation; Like the Bacillus bacterium: bacillus licheniformis (Bacillus licheniformis); Bacillus megatherium (Bacillus megaterium), bacillus brevis (Bacillus brevis) and Tribactur (Bacillus thuringiensis) or the like, these bacteriums also be widely used in industrial production albumen field [25].In addition, cytoplasm protein (being artificial secretory protein) after signal peptide is added in manual work also can be discerned by the bacterium excretory system, and then it is secreted into [26,34] in the substratum.Because the artificial secretory protein that makes up does not often pass through signal peptide optimization, thereby this proteinoid signal peptide efficient in Bacillus subtillis is often also lower.The target approach also can increase the efficient that the host secretes artificial secretory protein after the translation of the SecB mediation that the present invention relates in theory.Therefore, the present invention demonstrates wide application prospect.

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Claims (19)

1. the method for target approach after the translation that in bacterium, makes up the SecB mediation is characterized in that this method comprises:

Chimeric SecA albumen of coexpression and SecB albumen in this bacterium, thereby target approach after the translation that structure SecB mediates in this host;

Wherein, the sequence allos of said chimeric SecA proteic " zinc-binding motif " and this proteic other part, and said chimeric SecA albumen can combine said SecB albumen.

2. a method that increases bacterium secretory protein efficient is characterized in that, this method comprises:

Chimeric SecA albumen of coexpression and SecB albumen in this bacterium, thus in this host, make up target approach after the translation of SecB mediation, increase the efficient of this bacterium secretory protein thus;

Wherein, the sequence allos of said chimeric SecA proteic " zinc-binding motif " and this proteic other part, and said chimeric SecA albumen can combine said SecB albumen.

3. a method that improves the bacterioprotein secretion capacity is characterized in that, this method comprises:

Chimeric SecA albumen of coexpression and SecB albumen in this bacterium, thus target approach after the translation that structure SecB mediates in this host improves this protein of bacteria secretion capacity thus;

Wherein, the sequence allos of said chimeric SecA proteic " zinc-binding motif " and this proteic other part, and said chimeric SecA albumen can combine said SecB albumen.

4. like each described method in the claim 2～3, it is characterized in that said albumen is selected from natural secretory protein or artificial secretory protein, wherein natural secretory protein is preferably the natural secretory protein of external source.

5. method as claimed in claim 4 is characterized in that, the natural secretory protein of said external source is selected from lytic enzyme or pharmaceutical albumen, is preferably proteolytic enzyme, glycase, lypase, antibody, Interferon, rabbit, growth factor.

6. like each described method in the claim 4～5, it is characterized in that said albumen is fusion rotein, be preferably with maltose binding protein and merge and the fusion rotein of formation.

7. like each described method in the claim 1～6, it is characterized in that said bacterium is the bacterium of natural disappearance secB gene, and the natural secA gene that exists of said bacterium.

8. like each described method in the claim 1～7; It is characterized in that said bacterium is for being selected from the bacterium of Bacillus (Bacillus), Corynebacterium (Corynebacterium), mycobacterium (Mycobacterium), streptomyces (Streptomyces), Staphylococcus (Staphylococcus), lactobacillus (Lactobacillus), streptococcus (Streptococcus) or fusobacterium (Clostridium).

9. like each described method in the claim 1～8; It is characterized in that said bacterium is selected from subtilis (Bacillus subtilis), bacillus licheniformis (Bacillus licheniformis), Bacillus megatherium (Bacillus megaterium), bacillus brevis (Bacillus brevis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus pumilus (Bacillus pumilus) or Tribactur (Bacillus thuringiensis).

10. like each described method in the claim 1～9, it is characterized in that, said in this bacterium the chimeric SecA albumen of coexpression and SecB albumen comprise:

Structure contains chimeric secA expression carrier and contains the secB expression carrier; Wherein, Said chimeric secA gene is that artificial reconstructed said bacterium secA gene gets; The encoding sequence that is said bacterium secA gene " zinc-binding motif " is replaced by the encoding sequence of external source secA gene " zinc-binding motif ", thereby possesses the proteic ability of the said SecB of combination;

Contain chimeric secA expression carrier and contain the secB expression carrier and transform this bacterium with said, thus in this bacterium chimeric SecA albumen of coexpression and SecB albumen.

11. method as claimed in claim 10 is characterized in that, said secB gene comes from the bacterium with said bacterium different genera.

12., it is characterized in that said secB gene and the encoding sequence of " zinc-binding motif " of said secA gene derive from the bacterium of identical kind like each described method in the claim 10～11.

13., it is characterized in that " zinc-binding motif " of described external source secA gene is terminal last 18～60 amino acid of external source SecA protein carboxyl groups like each described method in the claim 10～12.

14., it is characterized in that said external source secA gene is intestinal bacteria secA gene or hemophilus influenzae secA gene like each described method in the claim 10～13.

15., it is characterized in that said external source secB gene is intestinal bacteria secB gene or hemophilus influenzae secB gene like each described method in the claim 10～14.

16. an aminoacid sequence is characterized in that, this aminoacid sequence contains SEQ ID NO:29 or the described aminoacid sequence of SEQ ID NO:31.

17. a nucleotide sequence is characterized in that, the described aminoacid sequence of said nucleotide sequence coded claim 16.

18. a construction is characterized in that, said construction contains the described nucleotide sequence of claim 17.

19. a system is characterized in that, said system contains:

(a) chimeric secA gene or contain the construction of this chimeric secA gene; Or the chimeric SecA albumen of said chimeric secA coded by said gene; Wherein, Other part allos of the encoding sequence of " zinc-binding motif " of said chimeric secA gene and this chimeric secA gene, chimeric SecA albumen possess the proteic ability of the said SecB of combination; With

(b) secB gene or contain the construction of this chimeric secB gene, or the SecB albumen of said secB coded by said gene possess the protein bound characteristic by said chimeric SecA.

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