CN102533627B - Functional construction and application of SecB-mediated translated targeted path - Google Patents
Functional construction and application of SecB-mediated translated targeted path Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C07K2319/00—Fusion polypeptide
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
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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
Technical field
The invention belongs to gene engineering technology field, relate to chimeric SecA and based on chimeric SecA, this technological method of the structure of target approach after the translation being mediated by SecB, and the application of this technological method in industrial production secretory protein.
Background technology
Bacterium is widely used for large scale fermentation and produces the albumen with various using values, as: pharmaceutical protein, industrial enzymes etc.At present, according to target protein after fermentation ends, be arranged in tenuigenin or substratum, production technology can be divided into two classes: a) with the mode productive target albumen of intracellular protein; 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, after fermentation ends, need not reclaim cell, need not take physics, chemical or biological method by lysis so that the release of target protein, therefore greatly simplify the separation and purification work of target protein after fermentation, effectively controlled production cost [1].In addition, with the mode productive target albumen of extracellular protein, also there is other advantage, for example: can effectively reduce the accumulation of tenuigenin internal object albumen, thereby avoid the formation [2] of occlusion body; The albumen that allows to contain disulfide linkage can carry out correct folding etc. under oxidative environment.Therefore at the industrial mode large scale fermentation productive target albumen with secretory protein, be, preferred option.
Bacterium can be tenuigenin, to be transported in the substratum of extracellular from its translation place by 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, and its core forms [3] by transmembrane protein passage SecYEG and molecular motor SecA (ATP enzyme), as shown in Figure 5.The new polypeptide chain that carries signal peptide need to be transferred on the key ingredient SecA of Sec translocase from tenuigenin, and this process is called target; Subsequently, in the situation that hydrolysising ATP provides energy, SecA squeezes out tenuigenin by new polypeptide chain to be secreted via SecYEG albumen passage, thereby enters the cytolemma external space [4].Up to the present, as shown in Figure 5, the approach of the mediated targeted process of having identified in bacterium has: 1) the common translation target approach of signal peptide identification particle (SRP) and acceptor (SR) mediation thereof, be responsible for the target of the secretory protein that newborn signal peptide hydrophobicity is strong, it is the signal peptide that SRP identification is exposed to rrna surface, in translation, by the interaction of SRP and its acceptor SR, the peptide chain in translation is transferred to [5] on Sec translocase, 2) target approach after the common translation/translation of SecA mediation, the target of the secretory protein that responsible signal peptide hydrophobicity is general, SecA need to be by the newborn signal peptide [6] of other factor Direct Recognition, 3) target approach after the translation of SecB mediation, assist SecA to identify (the i.e. secreted system identification of newborn secretion peptide chain of those signal peptide efficiency, in conjunction with and be transported to the efficiency in cytolemma outside, influence factor comprises signal peptide characteristic and signal peptide downstream peptide section characteristic) target of low secretory protein, thereby newborn secretion peptide chain is combined with SecB and is maintained non-folding conformation in translation latter stage or after finishing, the high-affinity of this binary complex and then dependence SecB and Sec translocase key ingredient SecA (by " electronegative region " and SecA dimer C-terminal " positively charged zinc structural domain " i.e. interaction between " zinc-binding motif " mediation on SecB tetramer surface), thereby peptide chain is transferred to SecA, complete 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 famous with its powerful protein excretion ability, can be by albumen direct secretion to the level [11,12] that makes it to reach gram every liter in substratum.This characteristic is produced highly beneficial for industrial enzymes, therefore this bacterioid is widely used at industrial production related products [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 the oneself protein of natural secretion, as: amylase and proteolytic enzyme [2].Although be no lack of examples of many successful with Bacillus subtillis production external source secretory protein, yet compare with endogenous secretory protein, the efficiency of in most cases Bacillus subtillis secretion foreign protein is still low, especially the albumen that those eukaryotes are originated, this layer of meaning, this has limited its widespread use [15,16] in industry.Research shows, the factor of restriction heterologous protein secretion efficiency is mainly target efficiency and proteasome degradation, Given this, researchist has been developed various strategies to strengthen the secretion capacity of Bacillus subtillis to foreign protein, for example: optimize signal peptide, cross expression targeting factor (SRP), cross expression Sec translocase component, cross the bacterial strain of expressing mate molecule and using Deficient In Extracellular Proteases etc., and these strategies have also been applied 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 better specific living environment, protein secreting system based on Sec translocase (being called for short Sec system) presents certain difference in the process of evolving between different species, such as: the difference [18] of the difference of signal peptide characteristic [17] and Sec system etc.The existence of these differences has caused external source secretory protein as endogenous secretory protein, in heterologous host, efficiently to be secreted just.If can overcome these differences, be expected to fundamentally solve the low problem of foreign protein secernment efficiency in heterologous host.The result of having delivered shows, compares with intestinal bacteria, and Bacillus subtillis has lacked target approach [18] after the translation of SecB mediation.Yet than intestinal bacteria signal peptide hydrophobicity, stronger and N end is with more positive charge [17] due to its signal peptide, so endogenous secretory protein can effectively be identified by SRP [19].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 cannot effectively be identified by SRP or SecA in Bacillus subtillis, cause secretory protein precursor to accumulate in tenuigenin and/or cause protein quality Controlling System and degraded, the target inefficiency that causes these foreign proteins, finally cause secernment efficiency low [20,21].In order to impel Bacillus subtillis at the industrial secretion production foreign protein that is widely used in, must solve the problem of target inefficiency.A way is transformation signal peptide or screening Optimal Signals peptide [22-24], thereby obtains being applicable to the signal peptide of target protein.Because specific target protein needs specific signal peptide, just can reach desirable target efficiency, a shortcoming of this strategy is exactly for different target proteins, need to do specific signal peptide transformation or screening.Based on the understanding to protein secreting on molecular level, consider 1) 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, therefore the present invention attempts target approach after the translation of in Bacillus subtillis reconstruct SecB mediation, to strengthening the secretion capacity of this bacterium to external source secretory protein, the maltose binding protein mutant (Ma1E11) of originating with intestinal bacteria, the fusion rotein (MalE-PhoA) of alkaline phosphatase (PhoA) and wild-type maltose binding protein and alkaline phosphatase is as 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 external source secretory protein, completed thus the present invention.
Summary of the invention
The invention provides a kind of method that increases bacterium secretory protein efficiency, the method comprises:
The chimeric SecA albumen of coexpression and SecB albumen in this bacterium, thus in this host, build target approach after the translation of SecB mediation, increase thus the efficiency of this bacterium secretory protein;
Wherein, the sequence allos of " zinc-binding motif " of described chimeric SecA albumen and the other parts of this albumen, and described chimeric SecA albumen can be in conjunction with described SecB albumen.
The method that the invention provides target approach after a kind of translation that builds SecB mediation in bacterium, the method comprises:
The chimeric SecA albumen of coexpression and SecB albumen in this bacterium, thus in this host, build target approach after the translation of SecB mediation;
Wherein, the sequence allos of " zinc-binding motif " of described chimeric SecA albumen and the other parts of this albumen, and described chimeric SecA albumen can be in conjunction with described SecB albumen.
The invention provides a kind of method that improves bacterioprotein secretion capacity, the method comprises:
The chimeric SecA albumen of coexpression and SecB albumen in this bacterium, thus in this host, build target approach after the translation of SecB mediation, improve thus the protein secreting ability of this bacterium;
Wherein, the sequence allos of " zinc-binding motif " of described chimeric SecA albumen and the other parts of this albumen, and described chimeric SecA albumen can be in conjunction with described SecB albumen.
In one embodiment, described 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, described albumen is selected from the natural secretory protein of external source, comprises lytic enzyme (for example: proteolytic enzyme, amylase or lipase), antibody, Interferon, rabbit and somatomedin.
In one embodiment, described albumen is fusion rotein, is preferably the fusion rotein that merges with maltose binding protein and form.
In one embodiment, described bacterium is the bacterium of natural disappearance secB gene, and the natural secA gene that exists of described bacterium.
In one embodiment, described 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, described 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, described in this bacterium the chimeric SecA albumen of coexpression and SecB albumen comprise:
Build the expression vector that contains chimeric secA gene and the expression vector that contains secB gene, wherein, described chimeric secA gene is that artificial reconstructed described bacterium secA gene obtains, the encoding sequence that is described bacterium secA gene " zinc-binding motif " is replaced by the encoding sequence of external source secA gene " zinc-binding motif ", thereby possesses the ability in conjunction with described SecB albumen;
With described expression vector containing chimeric secA gene with containing the expression vector of secB gene, transform this bacterium, thus in this bacterium the chimeric SecA albumen of coexpression and SecB albumen.
In one embodiment, described secB gene comes from the bacterium with described bacterium different genera.
In one embodiment, described secB gene and the encoding sequence of " zinc-binding motif " of described secA gene derive from the bacterium of identical kind.
In one embodiment, " zinc-binding motif " of described external source secA coded by said gene SecA albumen is last 18～60 amino acid of external source SecA protein carboxyl groups end.
In one embodiment, described external source secA gene is intestinal bacteria secA gene or hemophilus influenzae secA gene.
In one embodiment, described external source secB gene is intestinal bacteria secB gene or hemophilus influenzae secB gene.
The invention provides a kind of aminoacid sequence, this aminoacid sequence contains the aminoacid sequence described in SEQ ID NO:29 or SEQ ID NO:31.
The invention provides a kind of nucleotide sequence, described nucleotide sequence coded power aminoacid sequence of the present invention.
The invention provides a kind of construction, described construction is containing nucleotide sequence of the present invention.
The invention provides a kind of system, described system contains:
(a) chimeric secA gene or containing the construction of this chimeric secA gene, or the chimeric SecA albumen of described chimeric secA coded by said gene, wherein, the encoding sequence of " zinc-binding motif " of described chimeric secA gene and the other parts allos of this chimeric secA gene, chimeric SecA albumen possesses the ability in conjunction with described SecB albumen; With
(b) secB gene or containing the construction of this chimeric secB gene, or the SecB albumen of described secB coded by said gene, possess the protein bound characteristic by described chimeric SecA.
Accompanying drawing explanation
Fig. 1: pSJ3-ecSecA plasmid schematic diagram.
Fig. 2: pMA5-ecMalE11 plasmid schematic diagram.
Fig. 3: pAX01-ecSecB plasmid schematic diagram.
Fig. 4: pOE-beSecA plasmid schematic diagram.
Fig. 5: Bacillus subtillis protein target approach schematic diagram.Semi-circular dash area be the present invention relates to based on chimeric SecA, target approach after the translation being mediated by SecB.
Fig. 6: SecA protein carboxyl groups end determines that SecA-SecB specificity interacts.
Fig. 7: the chimeric SecA albumen of coexpression (beSecA) can strengthen with SecB albumen (ecSecB) ability that Bacillus subtillis secretes foreign protein MalE11.
Target approach after the translation of the efficient secretion dependence SecB mediation of Fig. 8: MalE11.
After the translation of Fig. 9: SecB mediation, target approach can strengthen the ability of Bacillus subtillis secretion foreign protein PhoA and MalE-PhoA fusion rotein.
Figure 10: " zinc-binding motif " of SecA protein carboxyl groups end determines that SecA-SecB specificity interacts.
Embodiment
In this application, described " 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 " mainly refer to that its wild-type contains SecA albumen but the bacterium of disappearance SecB albumen.More preferably, " host " of the present invention or " protein production bacterium " or " bacterium " are mainly gram positive bacteriums, comprise that those come from Bacillus (Bacillus), Corynebacterium (Corynebacterium), mycobacterium (Mycobacterium), streptomyces (Streptomyces), Staphylococcus (Staphylococcus), lactobacillus (Lactobacillus), streptococcus (Streptococcus), the bacterium of fusobacterium (Clostridium) or other genus, be specially subtilis (Bacillus subtilis), bacillus licheniformis (Bacillus licbeniformis), Bacillus megatherium (Bacillus megaterium), bacillus brevis (Bacillus brevis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus pumilus (Bacillus pumilus), the bacterium of Tribactur (Bacillus thuringiensis) or other kinds.
In the application, " external source ", is for endogenous, refers to that certain component derives from the bacterium of host described in 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 ability and the efficiency of external source secretory protein are improved to make to produce 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 albumen (being endogenous secretory protein) of the natural secretion of Host Strains or protein production bacterium self, natural secretory protein (being external source secretory protein) with source of species outside this Host Strains or protein production bacterium, the signal peptide that these albumen carry can be wild-type, can be also the signal peptide obtaining after artificial replacement or sudden change wild-type signal peptide.
Except these natural secretory proteins, can adopt the albumen of the application's method production can be also artificial constructed secretory protein, " the artificial secretory protein " that natural non-secretory protein (natural no signal peptide) obtains after utilizing genetic engineering technique manually to add signal peptide.Non-secretory protein can be both the albumen of the natural production of Host Strains self, the albumen that also the species outside Host Strains are produced.
In addition, can adopt the albumen of the application's method production can be fusion rotein.For example, described endogenous secretory protein, external source secretory protein and artificial secretory protein can be with form expression and secretions in Host Strains of the present invention or protein production bacterium of various fusion roteins.The expression vector that can contain this endogenous secretory protein, external source secretory protein or artificial secretory protein and the encoding sequence of the fusion rotein of other albumen formation by structure, then this carrier is proceeded in Host Strains or protein production bacterium and carries out expression and secretion.In a preferred embodiment, the preferred fusion rotein of above-mentioned endogenous or external source secretory protein, natural or artificial secretory protein and maltose binding protein formation.The method that builds and transform is all this area routine.
In a preferred embodiment, can adopt the albumen of method of the present invention or bacterium secretion to comprise that various industrial enzymes (for example: proteolytic enzyme, amylase, lipase etc.) and various pharmaceutical albumen (for example antibody, Interferon, rabbit, somatomedin etc.) [see 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, secreted.
The method that the present invention transforms bacterium includes but not limited to: build the expression vector that contains chimeric secA gene and the expression vector that contains SecB gene, then with described expression vector containing chimeric secA gene with containing the expression vector of secB gene, transform this bacterium, thus in this bacterium the chimeric SecA albumen of coexpression and SecB albumen.
In the present invention, chimeric secA gene is that artificial reconstructed Host Strains secA gene obtains, the encoding sequence that is described bacterium secA gene " zinc-binding motif " is replaced by the encoding sequence of external source secA gene " zinc-binding motif ", thereby possesses the ability in conjunction with described SecB albumen.
Herein, SecA albumen " zinc-binding motif " refers to the CXCX8C (C/X) of SecA protein carboxyl groups (C) end and the conservative amino acid residues closing on thereof, be responsible for the interaction of mediation and SecB albumen, should " zinc-binding motif " be generally arranged in last approximately 40 amino acid residue sequences of SecA protein carboxyl groups end.Can use " zinc-binding motif " of SecA albumen of the bacterium of the target approach that carrys out self-contained SecB mediation to replace " zinc-binding motif " of host or protein production bacterium SecA albumen.As long as the region being replaced comprises " zinc-binding motif ", the region of replacement can be " zinc-binding motif ", can be also the region of more growing, approximately 60,55,50,45 amino acid or shorter as last in SecA PROTEIN C end.Therefore, in certain embodiments, for example, with last 18～60 amino acid of external source SecA PROTEIN C end (, last 18～40,20～35,22～35,22～32 amino acid), replace the corresponding section of host or protein production bacterium SecA PROTEIN C end.In other embodiments, for the sequence of replacing, not necessarily from last amino acid of this external source SecA PROTEIN C end, start at.For example, the sequence that is used for replacing can be this external source SecA PROTEIN C end 2nd～40 amino acids, 2nd～35 amino acids, 2nd～32 amino acids, 3rd～40 amino acids, 3rd～35 amino acids etc. and the arbitrary amino acid fragment within the scope of these, as long as sequence or the amino acid fragment replaced still retain the biological function of " zinc-binding motif ".The chimeric SecA that the method builds possesses the ability in conjunction with the SecB albumen with its " zinc-binding motif " homology.For example, can example " zinc-binding motif " from bacterium SecA albumen such as intestinal bacteria, hemophilus influenzae, A.Tumefaciens, P.fluorescens, R.etli, A.Pleuropneumoniae as shown in Figure 6A replace " zinc-binding motif " of host or protein production bacterium SecA albumen.In a specific embodiment, can use the sequence from bacteriums such as intestinal bacteria, hemophilus influenzae, A.Tumefaciens, P.fluorescens, R.etli, A.Pleuropneumoniae that Fig. 6 A specifically lists to replace " zinc-binding motif " of SecA albumen in Host Strains.
Can adopt the method for this area routine to carry out above-mentioned replacement as merged PCR.For example, the described structure of the embodiment of the present application 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 the known material in this area (Bacillus subtillis expression vector) and technology (PCR and molecular cloning) to build the expression vector that the present invention contains the expression vector of chimeric secA gene and contains external source secB gene.Adopt subsequently and well known in the artly such as chemical conversion process, affiliated expression vector is incorporated in Bacillus subtillis cell.Being applicable to expression vector of the present invention can be integrated expression vector and rf expression vector, and the former is as pAX01, pA-spac or pDG1661 etc.; The latter is as pUB110 series derivatives plasmid (pMA5 or pWB980) or pBS72 series derivatives plasmid (pHCMC05 or pOE) etc.In addition the visible reference of a large amount of operable expression vectors [14].
Whether the bacterium that can adopt this area ordinary method to detect to be converted chimeric SecA albumen and external source SecB albumen described in stably express.These methods comprise SDS-PAGE and the immune marking subsequently, as described embodiments.
Herein, secB gene or its proteins encoded preferably derive from the encoding sequence that builds chimeric secA gene " zinc-binding motif " used but are not limited to same species or nearly source species, and the standard of selection is that selected SecB must carry out functional interaction in vivo with chimeric SecA.
In a preferred implementation, the external source " zinc-binding motif " that is preferably used for building chimeric SecA albumen and this external source SecB homology, 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 protein excretion efficiency.
Herein, SEQ ID NO:28 has shown the nucleotide sequence of chimeric secA gene, and wherein 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 SEQID NO:31.The application also comprises a kind of nucleotide sequence, and this sequence encoding the application is containing the aminoacid sequence of the sequence shown in SEQ ID NO:29 or SEQ ID NO:31.The application also comprises the construction that contains nucleotide sequence described in the application.In a preferred embodiment, described construction can be a kind of carrier.In preferred embodiment, described construction can be a kind of expression vector, for the chimeric SecA albumen of expressing the application host or protein production bacterium.Can adopt the technique means of this area routine to build the expression vector that contains described Nucleotide.
The application also comprises the purposes of above-mentioned aminoacid sequence, nucleotide sequence and construction, for example, for increasing the efficiency of protein production bacterium secretion foreign protein, for improving the production of external source secretory protein and comparing protein production bacterium that its foreign protein throughput is improved etc. for building to contrast with its wild-type.
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, described system is a kind of cell or bacterium, telescoping part in described chimeric secA gene (for example: the encoding sequence of " zinc-binding motif "), with described secB gene for this cell or bacterium, be all external source, and the telescoping part in described chimeric SecA albumen and SecB albumen are all also external sources for this cell or bacterium.
In a specific embodiment, the nucleotide sequence that described system contains intestinal bacteria secB gene described in nucleotide sequence described in SEQ ID NO:28 or 30 and GenBank:M24489.1, or the aminoacid sequence shown in SEQ ID NO:29 or 31 and the coded aminoacid sequence of GenBank:M24489.1.In a specific embodiment, described system is Bacillus subtillis.In other embodiments, described 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 the mode with specific embodiment is described in detail the present invention.Should be understood that these embodiment are only illustrative.In addition, herein, the terms such as " containing ", " comprising " also comprised in this article " by ... form ", " by ... form " etc. implication.The experimental technique of unreceipted actual conditions in the following example, conventionally according to routine operation, carry out the < < molecular cloning of for example publishing with reference to press of cold spring harbor laboratory: the laboratory manual > > third edition or the condition of advising according to article manufacturer used.
One. materials and methods
1) bacterial strain and plasmid
Plasmid pSJ2, pSJ3 and pSJ4 (document that sees reference [27]), be pET21a (Novagen) plasmid, at the recombinant expressed SecA of intestinal bacteria and SecB albumen.
Plasmid pMA5, provides [28] by Dartois, and kalamycin resistance, for expressing external source secretory protein Bacillus subtillis.
Plasmid pAX01, is provided by Bacillus subtilus DSMZ (BGSC), and erythromycin resistance, for expressing intestinal bacteria SecB albumen Bacillus subtillis.
Plasmid pOE, the pMD18 (TaKaRa) of take is basic framework, introduces and to come from pHCMC05[29] on Bacillus subtillis copy unit and chlorampenicol resistant mark; Introduce again the expression casette that HpaII promotor on pMA5 and colibacillary trpA terminator sequence form simultaneously.This plasmid is 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, take genome of E.coli as template, primer pair P1 (SEQ ID NO:1) and P2 (SEQ ID NO:2) amplification secA gene, after NdeI and BamHI double digestion, pack pSJ3 into, obtain pSJ3-ecSecA, its plasmid schematic diagram as shown in Figure 1.Plasmid pSJ3-bsSecA coding Bacillus subtillis SecA albumen, take Bacillus subtillis genome as template, primer pair P3 (SEQ ID NO:3) and P4 (SEQ ID NO:4) amplification secA gene, pack pSJ3 into after BamHI and XhoI double digestion, obtains pSJ3-bsSecA.
Plasmid pSJ3-bhSecA coding (Bacillus subtillis-hemophilus influenzae) chimeric SecA albumen is bhSecA, and this albumen is merged and obtained by the 1st, Bacillus subtillis SecA albumen to the 867th, the 809th amino acids and hemophilus influenzae SecA albumen to the 901st amino acids.This plasmid adopts large primer PCR technique construction, process is as follows: take hemophilus influenzae genome as template, the 867th encoding sequence to the 901st amino acids of primer pair P5 (SEQ ID NO:5) and P6 (SEQ ID NO:6) amplification hemophilus influenzae SecA albumen.Reclaim this segment as large primer, with P3 collocation, take plasmid pSJ3-bsSecA as template amplification obtains the encoding sequence of bhSecA albumen, after BamHI and HindIII double digestion, pack pSJ3 into, obtain pSJ3-bhSecA.In like manner, plasmid pSJ3-beSecA coding (Bacillus subtillis-intestinal bacteria) chimeric SecA is beSecA albumen, and this albumen is merged and obtained by the 1st, Bacillus subtillis SecA albumen to the 870th, the 809th amino acids and intestinal bacteria SecA albumen to the 901st amino acids.Take genome of E.coli as template, the 870th encoding sequence to the 901st amino acids of primer pair P7 (SEQ ID NO:7) and P2 amplification intestinal bacteria SecA albumen.Reclaim this segment as large primer, with P3 collocation, the plasmid pSJ3-bsSecA of take obtains the encoding sequence of beSecA albumen as template amplification, and BamHI enzyme packs pSJ3 into after cutting, and obtains pSJ3-beSecA.
B) pSJ4-hiSecB and pSJ2-ecSecB
Document [30] is shown in by the structure of plasmid pSJ4-hiSecB, this plasmid-encoded hemophilus influenzae SecB albumen.Plasmid pSJ2-ecSecB coding intestinal bacteria SecB albumen, take genome of E.coli as template, primer pair P8 (SEQ ID NO:8) and P9 (SEQ ID NO:9) amplification secB gene, BamHI and HindIII pair packs pSJ2 into after cutting, obtains pSJ2-ecSecB.
Above a) and b) in the plasmid that builds at the corresponding albumen of intestinal bacteria purifying, the albumen of these purifying is subsequently for external combination experiment and isothermal titration experiment.
C) pMA5-ecMalE11, pMA5-ecPhoA and pMA5-(ecMalE-PhoA)
The mutant of plasmid pMA5-ecMalE11 coding intestinal bacteria maltose binding proteins (MalE), i.e. MalE11 albumen, the N-terminal of this mutant carries 3 amino acid whose replacements (as shown in Figure 7 A).Sudden change is introduced by overlap extension pcr, plasmid construction process is as follows: take genome of E.coli as template, primer pair P10 (SEQ ID NO:10) and P11 (SEQ ID NO:11) amplification malE gene signal peptide coding region, primer pair P12 (SEQ ID NO:12) and P13 (SEQ ID NO:13) amplification malE gene mature peptide section coding region.The mixture of above-mentioned two purified PCR products of take is template, primer pair P10 and P13 amplification obtain the encoding sequence (sudden change is introduced by P10 and P12 respectively) of total length MalE11 albumen, after NdeI and HindIII double digestion, pack pMA5 into, obtain pMA5-ecMalE11, its plasmid schematic diagram as shown in Figure 2.
The plasmid pMA5-ecPhoA escherichia coli alkaline phosphatase (PhoA) of encoding, its building process is as follows: take genome of E.coli as template, primer pair P14 (SEQ ID NO:14) and P15 (SEQ ID NO:15) amplification phoA gene, after NdeI and HindIII double digestion, pack pMA5 into, obtain pMA5-ecPhoA.
The maltose binding protein in plasmid pMA5-(ecMalE-PhoA) coding intestinal bacteria source and the fusion rotein (MalE-PhoA) of alkaline phosphatase, the encoding sequence of this fusion rotein adopts overlap extension pcr to build.Plasmid construction process is as follows: take genome of E.coli as template, primer pair P16 (SEQ ID NO:16) does not contain the malE gene of terminator codon with P17 (SEQ ID NO:17) amplification; The encoding sequence in primer pair P18 (SEQ ID NO:18) and P15 amplification coding PhoA mature peptide section region.The mixture of above-mentioned two purified PCR products of take is template, and primer pair P16 and P15 amplification obtain the encoding sequence of total length MalE-PhoA albumen, after NdeI and HindIII double digestion, pack pMA5 into, obtain pMA5-(ecMalE-PhoA).
D) pAX01-ecSecB, pAX01-ecSecBL75Q, pAX01-ecSecBE77K and pAX01-ecSecBL75Q & E77K
Plasmid pAX01-ecSecB coding intestinal bacteria SecB albumen, take genome of E.coli as template, primer pair P19 (SEQ ID NO:19) and P20 (SEQ ID NO:20) amplification secB gene, BamHI enzyme packs pAX01 into after cutting, obtain pAX01-ecSecB, its plasmid schematic diagram as shown in Figure 3.
The mutant SecBL75Q of plasmid pAX01-ecSecBL75Q coding intestinal bacteria SecB albumen, sudden change is introduced by overlap extension pcr, plasmid construction process is as follows: take genome of E.coli as template, the segment of primer pair P19 and P21 (SEQ ID NO:21) amplification secB gene mutation site upstream, the segment in primer pair P22 (SEQ ID NO:22) and P20 amplification secB gene mutation site downstream, sudden change is introduced by P22.The mixture of above-mentioned two purified PCR products of take is template, and primer pair P19 and P20 amplification obtain the encoding sequence of total length SecBL75Q albumen, and BamHI enzyme packs pAX01 into after cutting, and obtains pAX01-ecSecBL75Q.With same method, P23 (SEQ ID NO:23) and P24 (SEQ ID NO:24) are respectively used to the structure of pAX01-ecSecBE77K and pAX01-ecSecBL75Q & E77K, these two plasmids encode respectively mutant SecBE77K and the SecBL75Q/E77K of intestinal bacteria SecB albumen.
E) pOE-beSecA and pOE-bsSecA.
Plasmid pOE-beSecA coding (Bacillus subtillis-intestinal bacteria) chimeric SecA is beSecA albumen, take plasmid pSJ3-beSecA as template, primer pair P25 (SEQ ID NO:25) increases and obtains the encoding sequence of beSecA albumen with P26 (SEQ ID NO:26), after KpnI and SacII double digestion, pack pOE into, obtain pOE-beSecA, its plasmid schematic diagram as shown in Figure 4.
Plasmid pOE-bsSecA coding Bacillus subtillis SecA albumen, take Bacillus subtillis genome as template, and primer pair P25 and P27 (SEQ ID NO:27) amplification secA gene, packs pOE into after KpnI and SacII double digestion, obtains pOE-bsSecA.
3) culture condition
If no special instructions, all adopt LB substratum, add suitable microbiotic, 37 degrees Celsius of shaking culture are spent the night.Antibiotic concentration is: penbritin 100ug/ml, kantlex 100ug/ml, paraxin 5ug/ml, erythromycin 5ug/ml.
4) method for transformation
Intestinal bacteria transform and adopt ripe calcium method to transform, and see the < < 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, the laboratory reference < < molecular cloning > > third editions such as Western Blotting.
6) alkaline phosphatase enzyme activity determination reference [32].
7) purifying of SecA albumen and SecB albumen and external combination experiment etc. are with reference to [27]
Two. result and discussion
Embodiment mono-
SecA protein carboxyl groups end determines that SecA-SecB specificity interacts.In the bacterium of disappearance secB gene, along with SecA protein carboxyl groups end " zinc-binding motif " the continuous accumulation of " detrimental mutation " during evolution, lost gradually the ability in conjunction with SecB albumen.By this class SecA protein carboxyl groups end " zinc-binding motif " replace with can with the corresponding section of the SecA albumen of SecB protein-interacting, can make originally can not obtain the ability in conjunction with SecB albumen in conjunction with the SecA albumen of SecB albumen, the Bacillus subtillis SecA of take is below explained as example.
Fig. 6 A shows the sequence comparison of the SecA albumen " zinc-binding motif " of different biogenetic derivations.As can be seen here, during evolution, although this motif presents high conservative property, sudden change is also significant.For the bacterium of those disappearances secB gene, as subtilis, sudden change may cause its SecA proteins lose in conjunction with the ability of SecB albumen.
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).While representing to build chimeric SecA, there is chimeric position in arrow indication part.
Fig. 6 C shows different SecA and the external combination situation of SecB, and result shows that bsSecA can not, in conjunction with hemophilus influenzae SecB (hiSecB), can not, in conjunction with intestinal bacteria SecB (ecSecB), be shown in swimming lane 1 and 2.This is consistent with the fact of Bacillus subtillis disappearance secB gene, and the continuous accumulation of " detrimental mutation " during evolution of its SecA protein carboxyl groups end " zinc-binding motif ", has lost the ability in conjunction with SecB albumen gradually.Yet, " zinc-binding motif " of bsSecA albumen replaced with to chimeric SecA that the corresponding section of hiSecA or ecSecA obtains is that bhSecA and beSecA have just obtained at least the ability in conjunction with the SecB albumen with its " zinc-binding motif " homology, see swimming lane 3,4 and 6.This result clearly illustrates that SecA protein carboxyl groups end determines that SecA-SecB specificity interacts, and can change it in conjunction with the characteristic of SecB by displacement SecA protein carboxyl groups end " zinc-binding motif ".
In order further to produce evidence, illustrate that chimeric SecA can be effectively in conjunction with SecB albumen, we have measured different SecA and the dissociation constant between ecSecB.Fig. 6 D demonstration, the dissociation constant between the dissociation constant between beSecA/ecSecB and positive control ecSecA/ecSecB is suitable, far below the dissociation constant between bsSecA/ecSecB.
Embodiment bis-
Based on chimeric SecA, can interact with external source SecB, in theory in the bacterium of secB genetically deficient the chimeric SecA albumen of coexpression and external source SecB albumen (both can interact) can reconstruct SecB the target approach of mediation, can strengthen the efficiency that host secretes foreign protein.Take that coexpression beSecA and ecSecB are as example is explained in Bacillus subtillis below, the external source secretory protein of selecting is MalE11.
MalE11 is the mutant of intestinal bacteria source MalE, the signal peptide n-quadrant of this mutant only with 1 clean positive charge (wild-type is 3 clean positive charges) and immediately the mature peptide section region of signal peptide also with 1 clean positive charge (wild-type net charge is 0), therefore called after MalE11, as shown in Figure 7 A, the secernment efficiency of this mutant in Bacillus subtillis compared and decreased with wild-type, therefore adopt this mutant to highlight the functional of target approach after the translation of SecB mediation in the present invention, strengthen the efficiency that host secretes foreign protein.
BsSecA or beSecA or empty carrier pOE will encode, tri-plasmids of pMA5 of coding ecSecB or empty carrier pAX01 and coding MalE11 are transformed in Bacillus subtillis simultaneously, obtain 6 strains and express respectively different SecA, the bacterial strain of ecSecB and MalE11 combination.These strain bacterium are cultivated after 15 hours in the LB substratum that adds 0.5% wood sugar induction ecSecB expression, sampling analysis, result as shown in Figure 7 B, single expression ecSecB, bsSecA or beSecA cannot increase the efficiency (the 2nd, 3 and 5 groups) that host secretes MalE11.The in the situation that of coexpression bsSecA and ecSecB (the 4th group), the secernment efficiency of MalE11 has a little raising, but the amplitude improving is weaker than the situation (the 6th group) of coexpression beSecA and ecSecB far away.The in the situation that of coexpression beSecA and ecSecB, the efficiency that host secretes MalE11 increases considerably, and the increase of MalE11 secretory volume is more than at least 1 times.We can see and in substratum, accumulated a large amount of MalE11 maturation proteins, and in tenuigenin, only accumulate a small amount of MalE11 precursor protein.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 efficiency that host secretes foreign protein.
Embodiment tri-
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 efficiency that host secretes foreign protein.
The expression of ecSecB is controlled by the promotor Pxy1 of wood sugar induction, in the experiment of this group, we regulate and control the expression level of ecSecB by adjusting the concentration of inductor wood sugar, 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 the impact of the expression level of investigation ecSecB on MalE11 secernment efficiency.Consistent with expection, Fig. 8 A clearly illustrates that, along with ecSecB expression level declines gradually, host secretes the efficiency of MalE11 and also successively decreases gradually, presents linear relationship.
Have more the experiment that convictive evidence comes from ecSecB mutant.The point mutation L75Q of SecB and E77K can destroy specificity between SecA-SecB and interact, but do not affect SecB, do not secrete the activity of peptide chain in conjunction with new life.Adopt these mutant, whether the efficient secretion that can investigate MalE11 relies on specificity between SecA-SecB interacts.EcSecB will encode, ecSecB L75Q, the pAX01 carrier of ecSecB E77K or ecSecB L75Q & E77K is transformed into respectively in the Bacillus subtillis of expressing beSecA and MalE11, under suitable condition incubated overnight, sampling analysis, result as shown in Figure 8 B.Compare with wild-type SecB (the 3rd group), SecB mutant (the 4th, 5 and 6 groups) can not be supported the efficient secretion of MalE11, illustrates that the efficient secretion of MalE11 relies on specificity interaction between SecA-SecB, and this result is in full accord with expection.
Embodiment tetra-
After the translation of SecB mediation, target approach can increase the efficiency that host secretes foreign protein and has certain versatility, and to be intestinal bacteria source alkaline phosphatase (PhoA) and maltose binding protein-alkaline phosphatase fusion protein (MalE-PhoA) be explained as example the external source secretory protein of take below.
Be similar to MalE11, Fig. 9 A has studied the secretory volume of PhoA under different SecA and ecSecB combination background in great detail, and in substratum, PhoA secretory volume can just be reflected by PhoA enzyme activity.The in the situation that of single expression ecSecB, the secretory volume of PhoA does not increase (comparing with No. 1 for No. 2).It should be noted that no matter be single expression bsSecA, or beSecA (comparing with No. 1 with No. 5 for No. 3), PhoA secretory volume all increases by 30% left and right, and this is consistent with bibliographical information, and SecA separately also can the newborn secretion of mediated targeted approach identification peptide chain.More it is worth noting, the in the situation that of beSecA and ecSecB coexpression, PhoA secretory volume has further increased again the secretory volume of PhoA increasing on the basis of 30% (expression by beSecA causes), final PhoA secretory volume cumulative rises more than 60%.As " reverse side " evidence, the L75Q sudden change of ecSecB interacts owing to having destroyed specificity between 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, the coexpression of above-mentioned these result proving again 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 efficiency that host secretes foreign protein.
Immunoblotting as shown in Figure 9 B, confirms that the coexpression of beSecA and ecSecB has increased the secretory volume of PhoA.Although its " apparent secernment efficiency " is very high, a large amount of PhoA precursor (swimming lane 2) in cellular component, do not detected.Contriver infers the in the situation that of coexpression beSecA and ecSecB, the target efficiency of PhoA increases greatly, avoided part PhoA precursor to be degraded in tenuigenin, thereby in the situation that the target approach of SecB mediation exists, the secretory volume of PhoA significantly increase.
The impact of the coexpression of Fig. 9 C demonstration beSecA and ecSecB on MalE-PhoA secernment efficiency.Immunoblotting confirms the in the situation that of beSecA and ecSecB coexpression, the secernment efficiency of MalE-PhoA is also greatly improved, in substratum, MalE-PhoA secretory volume significantly increases, and correspondingly the accumulation of its precursor in tenuigenin significantly reduces (swimming lane 3 and 4 contrasts 1 and 2).In addition, in substratum, enzyme activity determination has confirmed the result of the immune marking, and 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 telescoping part of the chimeric beSecA albumen of using in embodiment mono-to embodiment tetra-(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 upstream.Telescoping part (length can be less than 22 amino acid or be greater than 32 amino acid) is as long as comprise mediation and SecB interactional " zinc-binding motif " in theory, chimeric SecA just at least can be in conjunction with the ability of the SecB albumen with its " zinc-binding motif " homology, target approach after the translation of reconstruct SecB mediation in the bacterium that the coexpression of this chimeric SecA and this SecB can lack at secB, strengthen the efficiency that host secretes foreign protein, the bsSecA-R3 of take is below explained as example.
Figure 10 A shows that bsSecA-R3 compares with beSecA, only last 22 corresponding sections that amino acid substitution is ecSecA of bsSecA protein carboxyl groups end.Replace institute's calling sequence as shown in SEQ ID NO:31.
The in the situation that 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 host after the target approach enhancing host efficiency of secreting foreign protein.
Three. sum up
The present invention relates to structure and the application thereof of target approach after the translation of SecB mediation.Consider 1) Bacillus subtillis is at industrial production (endogenous) secretory protein that is widely used in; 2) because signal peptide characteristic presents significant difference between different biological groups, signal peptide inefficiency in Bacillus subtillis of external source secretory protein (as deriving from gram negative bacterium and Eukaryotic secretory protein); 3) after the translation of SecB mediation, target approach can effectively assist the new life of SecA recognition signal peptide inefficiency to secrete peptide chain; 4) Bacillus subtillis disappearance SecB albumen, therefore the present invention attempts target approach after the translation of in Bacillus subtillis reconstruct SecB mediation, to increasing the efficiency of this bacterium secretion external source secretory protein, further expand the application of this bacterium in industrial production secretory protein.Due to 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 built beSecA, it is the corresponding section that last 32 amino-acid residues of bsSecA protein carboxyl groups end are replaced into ecSecA albumen, this part comprises mediation SecA-SecB interactional " zinc-binding motif ", thereby obtains effective ability in conjunction with 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 mutant (MalE11) that the existence of this approach can effectively increase Bacillus subtillis secretion foreign protein, the efficiency of the fusion rotein (MalE-PhoA) of alkaline phosphatase (PhoA) and maltose binding protein and alkaline phosphatase, in substratum, the secretory volume of these albumen increases respectively more than 100%, 60% and 70%.In addition the functional of target approach after the translation of SecB mediation further verified in the control experiment be arrangeding in parallel.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 efficiency that host secretes foreign protein.In addition, technological method provided by the invention also can lack for other bacterium of target approach after the translation of SecB mediation, as Bacillus bacterium: bacillus licheniformis (Bacillus licheniformis), Bacillus megatherium (Bacillus megaterium), bacillus brevis (Bacillus brevis) and Tribactur (Bacillus thuringiensis) etc., these bacteriums be also widely used in industrial production albumen field [25].In addition, cytoplasm protein artificial add signal peptide after (being artificial secretory protein) also can be identified by bacterium excretory system, and then be secreted into [26,34] in substratum.Because the artificial secretory protein building does not often pass through signal peptide optimization, thereby this proteinoid signal peptide efficiency in Bacillus subtillis is often also lower.After the translation of the SecB mediation the present invention relates in theory,, target approach also can increase the efficiency that host secretes artificial secretory protein.Therefore, the present invention demonstrates wide application prospect.
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Claims (26)
1. a method that builds target approach after the translation of SecB mediation in bacterium, is characterized in that, the method comprises:
The chimeric SecA albumen of coexpression and SecB albumen in this bacterium, thus in this bacterium, build target approach after the translation of SecB mediation;
Wherein, described chimeric SecA albumen is selected from the albumen shown in SEQ ID NO:29 or 31, and described SecB albumen is intestinal bacteria SecB albumen; Or described chimeric SecA albumen merges and obtain to the 867th, the 809th amino acids and hemophilus influenzae SecA albumen to the 901st amino acids by the 1st, Bacillus subtillis SecA albumen, described SecB albumen is selected from intestinal bacteria SecB albumen or hemophilus influenzae SecB albumen;
Described chimeric SecA albumen can be in conjunction with described SecB albumen;
Described bacterium is subtilis (Bacillus subtilis), naturally has secA gene but natural disappearance secB gene; And
Described in this bacterium the chimeric SecA albumen of coexpression and SecB albumen comprise:
Build containing the expression vector of chimeric secA gene with containing the expression vector of secB gene, wherein, chimeric SecA albumen described in described chimeric secA genes encoding, SecB albumen described in described secB genes encoding; With
With described expression vector containing chimeric secA gene with containing the expression vector of secB gene, transform this bacterium, thus in this bacterium chimeric SecA albumen and SecB albumen described in coexpression.
2. the method for claim 1, is characterized in that, described secB gene and the encoding sequence of " zinc-binding motif " of described secA gene derive from the bacterium of identical kind.
3. the method for claim 1, is characterized in that, the aminoacid sequence of described chimeric SecA albumen is as shown in SEQ ID NO:29 or SEQ ID NO:31.
4. a method that increases bacterium secretory protein efficiency, is characterized in that, the method comprises:
The chimeric SecA albumen of coexpression and SecB albumen in this bacterium, thus in this host, build target approach after the translation of SecB mediation, increase thus the efficiency of this bacterium secretory protein;
Wherein, described chimeric SecA albumen is selected from the albumen shown in SEQ ID NO:29 or 31, and described SecB albumen is intestinal bacteria SecB albumen; Or described chimeric SecA albumen merges and obtain to the 867th, the 809th amino acids and hemophilus influenzae SecA albumen to the 901st amino acids by the 1st, Bacillus subtillis SecA albumen, described SecB albumen is selected from intestinal bacteria SecB albumen or hemophilus influenzae SecB albumen;
And described chimeric SecA albumen can be in conjunction with described SecB albumen;
Described bacterium is subtilis (Bacillus subtilis), naturally has secA gene but natural disappearance secB gene; And
Described in this bacterium the chimeric SecA albumen of coexpression and SecB albumen comprise:
Build containing the expression vector of chimeric secA gene with containing the expression vector of secB gene, wherein, chimeric SecA albumen described in described chimeric secA genes encoding, SecB albumen described in described secB genes encoding;
With described expression vector containing chimeric secA gene with containing the expression vector of secB gene, transform this bacterium, thus in this bacterium chimeric SecA albumen and SecB albumen described in coexpression.
5. method as claimed in claim 4, is characterized in that, described albumen is selected from natural secretory protein or artificial secretory protein.
6. method as claimed in claim 5, is characterized in that, the natural secretory protein that described natural secretory protein is external source.
7. method as claimed in claim 6, is characterized in that, the natural secretory protein of described external source is selected from lytic enzyme or pharmaceutical albumen.
8. method as claimed in claim 5, is characterized in that, described albumen is fusion rotein.
9. method as claimed in claim 8, is characterized in that, described fusion rotein is for merging with maltose binding protein the fusion rotein forming.
10. method as claimed in claim 4, is characterized in that, described secB gene and the encoding sequence of " zinc-binding motif " of described secA gene derive from the bacterium of identical kind.
11. methods as claimed in claim 4, is characterized in that, the aminoacid sequence of described chimeric SecA albumen is as shown in SEQ ID NO:29 or SEQ ID NO:31.
12. methods as claimed in claim 6, is characterized in that, the natural secretory protein of described external source is proteolytic enzyme, amylase, lipase, antibody, Interferon, rabbit or somatomedin.
13. improve a method for bacterioprotein secretion capacity, it is characterized in that, the method comprises:
The chimeric SecA albumen of coexpression and SecB albumen in this bacterium, thus in this host, build target approach after the translation of SecB mediation, improve thus the protein secreting ability of this bacterium;
Wherein, described chimeric SecA albumen is selected from the albumen shown in SEQ ID NO:29 or 31, and described SecB albumen is intestinal bacteria SecB albumen; Or described chimeric SecA albumen merges and obtain to the 867th, the 809th amino acids and hemophilus influenzae SecA albumen to the 901st amino acids by the 1st, Bacillus subtillis SecA albumen, described SecB albumen is selected from intestinal bacteria SecB albumen or hemophilus influenzae SecB albumen;
And described chimeric SecA albumen can be in conjunction with described SecB albumen;
Described bacterium is subtilis (Bacillus subtilis), naturally has secA gene but natural disappearance secB gene; And
Described in this bacterium the chimeric SecA albumen of coexpression and SecB albumen comprise:
Build containing the expression vector of chimeric secA gene with containing the expression vector of secB gene, wherein, chimeric SecA albumen described in described chimeric secA genes encoding, SecB albumen described in described secB genes encoding;
With described expression vector containing chimeric secA gene with containing the expression vector of secB gene, transform this bacterium, thus in this bacterium chimeric SecA albumen and SecB albumen described in coexpression.
14. method as claimed in claim 13, is characterized in that, described albumen is selected from natural secretory protein or artificial secretory protein.
15. method as claimed in claim 14, is characterized in that, the natural secretory protein that described natural secretory protein is external source.
16. methods as claimed in claim 15, is characterized in that, the natural secretory protein of described external source is selected from lytic enzyme or pharmaceutical albumen.
17. methods as claimed in claim 14, is characterized in that, described albumen is fusion rotein.
18. methods as claimed in claim 17, is characterized in that, described fusion rotein is for merging with maltose binding protein the fusion rotein forming.
19. methods as claimed in claim 13, is characterized in that, described secB gene and the encoding sequence of " zinc-binding motif " of described secA gene derive from the bacterium of identical kind.
20. methods as claimed in claim 13, is characterized in that, the aminoacid sequence of described chimeric SecA albumen is as shown in SEQ ID NO:29 or SEQ ID NO:31.
21. methods as claimed in claim 15, is characterized in that, the natural secretory protein of described external source is proteolytic enzyme, amylase, lipase, antibody, Interferon, rabbit or somatomedin.
22. 1 kinds of aminoacid sequences, it is characterized in that, this aminoacid sequence, as shown in SEQ ID NO:29 or SEQ ID NO:31, or is merged and is obtained by the 1st, Bacillus subtillis SecA albumen to the 867th, the 809th amino acids and hemophilus influenzae SecA albumen to the 901st amino acids.
23. a nucleotide sequence, is characterized in that, the aminoacid sequence described in described nucleotide sequence coded claim 22.
24. 1 kinds of constructions, is characterized in that, described construction is containing having the right to want the nucleotide sequence described in 23.
25. 1 kinds of bacteriums, is characterized in that, described bacterium contains:
(a) chimeric secA gene or containing the construction of this chimeric secA gene, or the chimeric SecA albumen of described chimeric secA coded by said gene, wherein, described chimeric SecA albumen is selected from the albumen shown in SEQ ID NO:29 or 31; With secB gene or containing the construction of this chimeric secB gene, or the SecB albumen of described secB coded by said gene, wherein, described SecB albumen is intestinal bacteria SecB albumen, described secB gene is intestinal bacteria secB gene; Or
(b) chimeric secA gene or containing the construction of this chimeric secA gene, or the chimeric SecA albumen of described chimeric secA coded by said gene, wherein, described chimeric SecA albumen is merged and is obtained by the 1st, Bacillus subtillis SecA albumen to the 867th, the 809th amino acids and hemophilus influenzae SecA albumen to the 901st amino acids; With secB gene or containing the construction of this chimeric secB gene, or the SecB albumen of described secB coded by said gene, wherein, described SecB albumen is selected from intestinal bacteria SecB albumen or hemophilus influenzae SecB albumen, and described secB gene is selected from intestinal bacteria secB gene or hemophilus influenzae secB gene;
Wherein, described chimeric SecA albumen possesses the ability in conjunction with described SecB albumen;
Described bacterium is subtilis (Bacillus subtilis), natural disappearance secB gene, and the natural secA gene that exists;
And wherein, this bacterium has ability and the efficiency of the production external source secretory protein of raising.
26. bacteriums as claimed in claim 25, is characterized in that, the aminoacid sequence of described chimeric SecA albumen is as shown in SEQ ID NO:29 or SEQ ID NO:31.
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- 2011-12-31 WO PCT/CN2011/085121 patent/WO2012089164A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101631862A (en) * | 2007-04-02 | 2010-01-20 | 花王株式会社 | Modified microorganism |
Non-Patent Citations (5)
| Title |
|---|
| JP特开2010-178714A 2010.08.19 |
| Structural determinants of SecB recognition by SecA in bacterial protein translocation;Zhou jiahai ＆Xu zhaohui;《Nature Structural Biology》;20031130;942-947 * |
| Zhou jiahai ＆Xu zhaohui.Structural determinants of SecB recognition by SecA in bacterial protein translocation.《Nature Structural Biology》.2003,942-947. |
| 分子伴侣过量表达对蛋白质分泌及可溶性的影响;杨运桂等;《中国生物化学与分子生物学报》;20000630;第16卷(第3期);382-387 * |
| 杨运桂等.分子伴侣过量表达对蛋白质分泌及可溶性的影响.《中国生物化学与分子生物学报》.2000,第16卷(第3期),382-387. |
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| WO2012089164A1 (en) | 2012-07-05 |
| CN102533627A (en) | 2012-07-04 |
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