WO2012089164A1 - Construction fonctionnelle d'une voie d'adressage post-traductionnel faisant intervenir secb et utilisation de cette construction - Google Patents

Construction fonctionnelle d'une voie d'adressage post-traductionnel faisant intervenir secb et utilisation de cette construction Download PDF

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WO2012089164A1
WO2012089164A1 PCT/CN2011/085121 CN2011085121W WO2012089164A1 WO 2012089164 A1 WO2012089164 A1 WO 2012089164A1 CN 2011085121 W CN2011085121 W CN 2011085121W WO 2012089164 A1 WO2012089164 A1 WO 2012089164A1
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protein
secb
gene
seca
chimeric
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PCT/CN2011/085121
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Chinese (zh)
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刁刘洋
周佳海
杨晟
罗兰佛洛德尔
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中国科学院上海生命科学研究院
中国科学院上海有机化学研究所
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Publication of WO2012089164A1 publication Critical patent/WO2012089164A1/fr

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    • C—CHEMISTRY; METALLURGY
    • 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)
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand

Definitions

  • the present invention belongs to the field of genetic engineering technology, and relates to a chimeric SecA and a chimeric SecA-based, SecB-mediated construction of a post-translational targeting pathway, and the use of the technical method for industrial production of secreted proteins. Background technique
  • Bacteria are widely used for large-scale fermentation to produce proteins of various applications, such as: pharmaceutical proteins, industrial enzymes, and the like.
  • the production technology can be divided into two categories: a) production of the target protein by intracellular protein; b) extracellular protein (ie, secreted protein) The way to produce the target protein.
  • the former has a target protein in the culture medium, and there is no need to recover cells after the fermentation, and it is not necessary to physically, chemically or biologically lyse the cells to release the target protein, thereby greatly simplifying the separation of the target protein after fermentation. Purification work effectively controls production costs [1].
  • the production of the target protein by extracellular protein has other advantages, such as: It can effectively reduce the accumulation of target proteins in the cytoplasm, thereby avoiding the formation of inclusion bodies [2]; allowing the protein containing disulfide bonds to be oxidative. Proper folding in the environment, etc. Therefore, large-scale fermentation production of target proteins in the form of secreted proteins in the industry is the preferred solution.
  • Bacteria are capable of transporting a protein containing a signal peptide (ie, a secreted protein) from its translation site, the cytoplasm, into an extracellular medium, a process known as protein secretion.
  • a signal peptide ie, a secreted protein
  • the implementation of this function is mainly dependent on Sec translocase, the core of which consists of the transmembrane protein channel SecYEG and the molecular motor SecA (ATPase) [3], as shown in Figure 5.
  • the nascent peptide chain carrying the signal peptide needs to be transferred from the cytoplasm to the key component SecA of the Sec transposase. This process is called targeting; subsequently, in the case of hydrolyzing ATP to provide energy, SecA will be secreted.
  • the peptide chain is extruded out of the cytoplasm via the SecYEG protein channel and enters the extracellular space of the cell [4].
  • the pathways that have been identified in bacteria to mediate targeted processes are: 1) signal peptide recognition particle (SRP) and its receptor (SR)-mediated co-translational targeting pathway, responsible for the targeting of secreted proteins with strong hydrophobicity of the nascent signal peptide, ie SRP recognizes the signal peptide exposed on the surface of the ribosome, and transfers the translated peptide chain to Sec by translation of SRP and its receptor SR.
  • SRP signal peptide recognition particle
  • SR receptor
  • Bacillus subtilis and its related Bacillus are known for their potent protein secretion capacity, allowing proteins to be secreted directly into the culture medium to levels of grams per liter [11, 12]. This property is very advantageous for the production of industrial enzymes, so these bacteria are widely used in the industrial production of related products [13, 14], for example: Among the existing commercial enzymes, about 60% belong to the leather Most of the natively secreted self-proteins, such as amylase and protease [2], are produced by the fermentation of Bacillus positive bacteria.
  • subtilis to secrete foreign proteins, such as: optimization of signal peptides, overexpression Targeting factors (SRP), overexpressing Sec transposase components, overexpressing chaperones, and using protease-deficient strains, etc., and these strategies have also been applied for corresponding patents [1].
  • SRP overexpression Targeting factors
  • Sec transposase components overexpressing chaperones
  • protease-deficient strains etc.
  • the Sec translocator-based protein secretion system shows some differences among different species during the evolution process, for example: Differences in signal peptide characteristics [17] ] and Sec system differences [18] and so on. It is the existence of these differences that result in the inability of exogenous secreted proteins to be efficiently secreted in heterologous hosts like endogenous secreted proteins. If these differences can be overcome, it is expected to fundamentally solve the problem of low secretion efficiency of foreign proteins in heterologous hosts. Published results indicate that B.
  • subtilis lacks a SecB-mediated post-translational targeting pathway compared to E. coli [18].
  • endogenous secreted proteins can be efficiently recognized by SRP because their signal peptides are more hydrophobic than E. coli signal peptides and have more positive charges at the N-terminus [17].
  • SRP secreted protein
  • subtilis resulting in Secreted protein precursors accumulate in the cytoplasm and/or trigger protein quality control systems to degrade them, resulting in low targeting efficiency of these foreign proteins, ultimately leading to low secretion efficiency [20, 21].
  • B. subtilis In order to promote the industrial use of B. subtilis for the secretion and production of foreign proteins, it is necessary to solve the problem of low targeting efficiency.
  • One approach is to engineer the signal peptide or screen for the optimal signal peptide [22-24] to obtain a signal peptide suitable for the protein of interest. Since a particular protein of interest requires a specific signal peptide to achieve the desired targeting efficiency, one drawback of this strategy is that specific signal peptide modifications or screens are required for different proteins of interest.
  • the present invention provides a method of increasing the efficiency of bacterial secreted proteins, the method comprising:
  • the chimeric SecA protein and the SecB protein are co-expressed in the bacterium, thereby constructing a SecB-mediated post-translational targeting pathway in the host, thereby increasing the efficiency of the bacterial secreted protein;
  • the "zinc binding motif" of the chimeric SecA protein is heterologous to the sequence of other portions of the protein, and the chimeric SecA protein is capable of binding to the SecB protein.
  • the present invention provides a method for constructing a SecB-mediated post-translational targeting pathway in bacteria, the method comprising: co-expressing a chimeric SecA protein and a SecB protein in the bacterium, thereby constructing a SecB-mediated translation in the host Targeting pathway
  • the "zinc binding motif" of the chimeric SecA protein is heterologous to the sequence of other portions of the protein, and the chimeric SecA protein is capable of binding to the SecB protein.
  • the invention provides a method for improving bacterial protein secretion ability, the method comprising:
  • the chimeric SecA protein and the SecB protein are co-expressed in the bacterium, thereby constructing a SecB-mediated post-translational targeting pathway in the host, thereby increasing the protein secretion ability of the bacterium;
  • the "zinc binding motif" of the chimeric SecA protein is heterologous to the sequence of other portions of the protein, and the chimeric SecA protein is capable of binding to the SecB protein.
  • the protein is selected from a natural secreted protein or an artificial secreted protein, wherein the native secreted protein is preferably a foreign native secreted protein.
  • the protein is selected from a foreign natural secreted protein, including a hydrolase (e.g., a protease, an amylase or a lipase), an antibody, an interferon, and a growth factor.
  • a hydrolase e.g., a protease, an amylase or a lipase
  • the protein is a fusion protein, preferably a fusion protein formed by fusion with a maltose binding protein.
  • the bacterium is a bacterium naturally deficient in the sec gene, and the bacterium naturally harbors the secA gene.
  • the bacterium is selected from the group consisting of Bacillus genus 03/4 ⁇ «, Corynebac terium, Mycobac terium, Strep tomyces, Staphylococcus ( Staphylococcus ), Lac tobacillus, Strep tococcus or Clostridium.
  • the bacterium is selected from the group consisting of Bacillus subtilis ⁇ Bacillus subtil is, Bacillus licheniformis, Bacillus mega terium, Bacillus brevis, Bacillus cerevisiae ⁇ Bacillus amyloliquefaciens ⁇ ), Bacillus pumilus or Bacillus thuringiensis.
  • the co-expression of the chimeric SecA protein and the SecB protein in the bacterium comprises: constructing an expression vector comprising a chimeric gene and an expression vector comprising the sec gene, wherein the chimeric gene is artificially transformed
  • the bacterial gene that is, the coding sequence of the bacterial gene "zinc binding motif” is replaced by the coding sequence of the foreign gene "zinc binding motif", thereby having the ability to bind the SecB protein;
  • the bacterium is transformed with the expression vector containing the chimeric gene and an expression vector containing the sec gene, thereby co-expressing the chimeric SecA protein and the SecB protein in the bacterium.
  • the sec gene is derived from a bacterium of a different species than the bacterium.
  • the coding sequence of the sec gene and the "zinc binding motif" of the gene is derived from a bacterium of the same species.
  • the "zinc binding motif" of the SecA protein encoded by the exogenous gene is the last 18 to 60 amino acids of the carboxy terminus of the exogenous SecA protein.
  • the exogenous gene is the Escherichia coli SGCA gene or the Haemophilus influenzae secA gene.
  • the exogenous set ⁇ gene is an E. coli set ⁇ gene or a Haemophilus influenzae ⁇ sec gene.
  • the present invention provides an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 31.
  • the present invention provides a nucleotide sequence encoding the amino acid sequence of the present invention.
  • the invention provides a construct comprising a nucleotide sequence of the invention.
  • the present invention provides a system comprising:
  • Figure 1 Schematic representation of the pSJ3-ecSecA plasmid.
  • Figure 2 Schematic representation of the pMA5-ecMalEll plasmid.
  • Figure 3 Schematic representation of the pAXOl-ecSecB plasmid.
  • Figure 4 Schematic representation of the pOE-Z1 ⁇ 2SecA plasmid.
  • FIG. 5 Schematic diagram of the B. subtilis protein targeting pathway.
  • the semicircular shaded portion is a post-translational targeting pathway mediated by SecB based on chimeric SecA of the present invention.
  • Figure 7 Co-expression of the chimeric SecA protein (Z ⁇ SecA) and the SecB protein (ecSecB) enhance the ability of B. subtilis to secrete the foreign protein MalEll.
  • Figure 8 The efficient secretion of MalEll is dependent on the SecB-mediated post-translational targeting pathway.
  • Figure 10 The "zinc binding motif" at the carboxy terminus of the SecA protein determines the SecA-SecB specific interaction. detailed description
  • the "host” or “protein-producing bacterium” or “bacteria” includes various protein-producing bacteria including Gram-positive bacteria and Gram-negative bacteria.
  • the "host” or “protein-producing bacterium” or “bacteria” of the present invention mainly refers to a bacterium having a wild-type SecA protein but lacking the SecB protein.
  • the "host” or “protein-producing bacteria” or “bacteria” of the present invention are mainly Gram-positive bacteria, including those from the genus ⁇ Bacillus, Corynebacterium, and Mycobacterium (Bacillus).
  • Mycobacterium Streptomyces, Staphylococcus, Lactobacillus, Streptococcus, Clostridium or other genus bacteria, specifically Bacillus subtilis (Bacillus) Subtilis), Bacillus licheniformis, sc ⁇ iAs 1 mega terium, sc ⁇ iAs 1 brevis, Bacillus amyloliquefaciens, Bacillus pumilus , Bacillus thuringiensis iBacillus thuringiensis) or other species of bacteria.
  • exogenous refers to a bacterium which is derived from a host or a protein-producing bacterium other than the present application, relative to an endogenous source, and mainly refers to a bacterium from a different species.
  • the above bacteria can be engineered to increase the ability and efficiency of producing secreted proteins, particularly exogenous secreted proteins.
  • Proteins produced by the methods of the present application include, but are not limited to, natural secreted proteins (naturally containing signal peptides), ie, proteins naturally secreted by the host or protein producing bacteria themselves (ie, endogenous secreted proteins), and production of the host bacteria or proteins.
  • the natural secreted proteins ie, exogenous secreted proteins
  • the signal peptides carried by these proteins may be wild type, or may be signal peptides obtained after artificial replacement or mutation of the wild type signal peptide.
  • the protein produced by the method of the present application may also be an artificially constructed secreted protein, that is, a natural non-secreted protein (natural signal-free peptide) obtained by artificially adding a signal peptide using genetic engineering techniques.
  • artificial secreted protein The non-secreted protein may be either a protein naturally produced by the host strain itself or a protein produced by a species other than the host strain.
  • the protein produced by the method of the present application may be a fusion protein.
  • the endogenous secreted protein, exogenous secreted protein and artificial secreted protein can be expressed and secreted in the form of various fusion proteins in the host or protein producing bacterium of the present invention.
  • the expression vector can be constructed by transferring an expression vector containing the coding sequence of the fusion protein of the endogenous secretory protein, exogenous secretory protein or artificial secreted protein with other proteins, and then transferring the vector into a host or protein producing bacterium for expression and secretion.
  • a fusion protein of the above-described endogenous or exogenous secreted protein, a natural or artificial secreted protein, and a maltose binding protein is preferred. Methods of construction and transformation are routine in the art.
  • the method of the present invention or the protein secreted by the bacteria may include various industrial enzymes (for example, proteases, amylases, lipases, etc.) and various medical proteins (eg, antibodies, interferons, growth). Factors, etc.) [See references 2, 25, 26].
  • industrial enzymes or pharmaceutical proteins may be secreted by the method or bacteria of the present invention in the form of a fusion protein (e.g., fused to a maltose binding protein).
  • the method for cultivating the bacterium of the present invention includes, but is not limited to, constructing an expression vector containing the chimeric gene and an expression vector containing the secB gene, and then transforming the bacterium with the expression vector containing the chimeric secA gene and the expression vector containing the secB gene.
  • the chimeric SecA protein and the SecB protein are co-expressed in the bacterium.
  • the chimeric gene is obtained by artificially modifying the host gene, that is, the coding sequence of the bacterial gene "zinc binding motif" is replaced by the coding sequence of the foreign gene "zinc binding motif", thereby having a binding site.
  • the ability of the SecB protein is obtained by artificially modifying the host gene, that is, the coding sequence of the bacterial gene "zinc binding motif" is replaced by the coding sequence of the foreign gene "zinc binding motif", thereby having a binding site. The ability of the SecB protein.
  • the SecA protein "zinc-binding motif” refers to the CXC C (C/X) at the carboxyl (C) terminus of the SecA protein and its adjacent conserved amino acid residues, which are responsible for mediating the interaction with the SecB protein.
  • the motif is generally located in the last about 40 amino acid residue sequences at the carboxy terminus of the SecA protein.
  • the "zinc binding motif" of the host or protein producing SecA protein can be replaced with a "zinc binding motif" of the SecA protein from a SecB-mediated targeting pathway.
  • the replaced region may include only a "zinc-binding motif", and the replaced region may be only a "zinc-binding motif” or a longer region, such as the C-terminus of the SecA protein, which is about 60, 55, 50, 45 at the end. Amino acid or shorter.
  • the C-terminal end of the exogenous SecA protein is used for the last 18 to 60 amino acids (eg, the last 18 to 40, 20 ⁇ ) 35, 22 to 35, 22 to 32 amino acids) Replace the corresponding portion of the C-terminus of the host or protein producing strain SecA protein.
  • the sequence used for substitution does not necessarily start from the last amino acid at the C-terminus of the exogenous SecA protein.
  • the sequence to be substituted may be the amino acids 2 to 40 of the C-terminus of the exogenous SecA protein, amino acids 2 to 35, amino acids 2 to 32, amino acids 3 to 40, amino acids 3 to 35. And any amino acid fragment within these ranges, as long as the substituted sequence or amino acid fragment retains the biological function of the "zinc binding motif".
  • the chimeric SecA constructed by this method possesses the ability to bind to a SecB protein homologous to its "zinc binding motif".
  • a host or protein producing bacterium can be replaced with a "zinc binding motif" such as the SecA protein of Escherichia coli, Haemophilus influenzae, A. Tumefaciens P. fluorescens, R.
  • etli A. Pleuropneumoniae, etc. as shown in Fig. 6A.
  • "Zinc-binding motif" of the SecA protein specifically from Escherichia coli, Haemophilus influenzae, A. T. faciens, P. fluorescens, R. etl A / e>i/r 0 /Me>TM 0 can be used as specifically illustrated in Figure 6A.
  • the sequence of bacteria such as 2iae> replaces the "zinc binding motif" of the SecA protein in the host strain.
  • substitutions can be carried out by methods conventional in the art such as fusion PCR.
  • a chimeric gene carrying an exogenous "zinc binding motif" coding sequence is constructed as described in the Examples section of the present application, and then the chimeric SecA protein is expressed in a host or protein producing bacterium.
  • the chimeric gene-containing expression vector and the exogenous sec gene-containing expression vector of the present invention can be constructed using materials well known in the art (B. subtilis expression vector) and techniques (PCR and molecular cloning).
  • the associated expression vector is then introduced into B. subtilis cells using methods well known in the art, such as chemical transformation methods.
  • the expression vector suitable for use in the present invention may be an integrative expression vector and a replication-type expression vector, such as pAXO1, pA-spac or pDG1661; the latter such as pUBl10 series-derived plasmid (pMA5 or pTO980) or pBS72 series-derived plasmid (pHCMC05) Or pOE) and so on.
  • pAXO1 pA-spac
  • pDG1661 pDG1661
  • pUBl10 series-derived plasmid pMA5 or pTO980
  • pHCMC05 pBS72 series-derived plasmi
  • Whether the transformed bacteria has stably expressed the chimeric SecA protein and the exogenous SecB protein can be detected by a conventional method in the art. These methods include SDS-PAGE and subsequent immunoblotting as described in the Examples.
  • the coding sequence of the sec gene or its encoded protein and the "zinc binding motif" used in the construction of the chimeric gene is preferably derived from, but not limited to, the same species or a near-source species, and the selection criteria is that the selected SecB must be selected. Functional interaction with chimeric SecA in vivo.
  • the exogenous "zinc binding motif" used to construct the chimeric SecA protein is homologous to the exogenous SecB, ie from the same bacteria, such as from Escherichia coli or influenza bloodthirsty. Bacillus.
  • the "zinc-binding motif" and SecB from different bacteria can ensure that the chimeric SecA and SecB can interact functionally in vivo, and can also function in the same host or protein-producing bacterium to increase protein secretion efficiency.
  • SEQ ID NO: 28 shows the nucleic acid sequence of the chimeric gene, wherein positions 2428 to 2526 are the corresponding portions of the E. coli gene.
  • SEQ ID NO: 29 shows the amino acid sequence of chimeric SecA.
  • SEQ ID NO: 30 shows the nucleic acid sequence of another chimeric gene of the present invention in which the phase of the E. coli secA gene is inserted.
  • SEQ ID NO: 31 shows the amino acid sequence thereof.
  • the application also includes an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 31.
  • the application also includes a nucleotide sequence encoding an amino acid sequence comprising the sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 31.
  • the present application also encompasses constructs comprising the nucleotide sequences described herein.
  • the construct can be a carrier.
  • the construct may be an expression vector for expression of the chimeric SecA protein of the present application in a host or protein producing bacterium. Expression vectors containing the nucleotides can be constructed using techniques routine in the art.
  • the present application also encompasses the use of the above amino acid sequences, nucleotide sequences and constructs, for example, for increasing the efficiency of secretion of foreign proteins by protein producing bacteria, for improving the production of exogenous secreted proteins, and for constructing with wild type A protein-producing bacterium that has an improved production capacity of the exogenous protein compared to the control.
  • the application also includes a system comprising a chimeric gene and gene as described herein, and/or a chimeric SecA protein and a SecB protein.
  • the system is a cell or a bacterium, a chimeric portion of the chimeric gene (eg, a coding sequence for a "zinc binding motif"), relative to the sec gene
  • a chimeric portion of the chimeric gene eg, a coding sequence for a "zinc binding motif”
  • the system comprises the nucleotide sequence set forth in SEQ ID NO: 28 or 30 and the nucleotide sequence of the E. coli set ⁇ gene of GenBank: M24489. 1, or SEQ ID NO: 29 Or the amino acid sequence shown by 31 and the amino acid sequence encoded by GenBank: M24489.
  • the system is Bacillus subtilis.
  • the system is Bacillus licheniformis, bacillus mega terium, bacillus bre vis, Bacillus amyloliquefaciens, Bacillus pumilus ) or Bacillus thuringiensis and the like.
  • Plasmids pSJ2, pSJ3 and pSJ4 are both of which are pET21a ( Novagen)-derived plasmids for recombinant expression of SecA and SecB proteins in E. coli.
  • the plasmid pAXO1 supplied by the Bacillus subtilis collection (BGSC), is erythromycin resistant and is used to express E. coli SecB protein in B. subtilis.
  • Plasmid pOE using pMD18 (TaKaRa) as the basic framework, introduced Bacillus subtilis replicator and chloramphenicol resistance marker from pHCMC05 [29]; simultaneously introduced Hpall promoter on PMA5 and trpA termination sequence of E. coli A gene expression cassette consisting of. This plasmid was used to express the SecA protein in B. subtilis.
  • the cloning host E. coli DH5a and the protein expression host E. coli BL21 (DE3) were supplied by Novagen.
  • Bacillus subtilis 168 ⁇ Bacillus subtil is 168) is provided by the Bacillus subtilis collection (BGSC).
  • Plasmid pSJ3-e> C S ec A encodes E. coli SecA protein, using E. coli genome as template, primer pair PKSEQ ID N0:1) and P2 (SEQ ID NO: 2) amplification gene, Ndel and BamHI after double digestion
  • the pSJ3 was loaded to obtain pSJ3-e> C Se C A, and the plasmid diagram is shown in Fig. 1.
  • the plasmid pSJ3-feSe C A encodes the Bacillus subtilis SecA protein, using the B.
  • subtilis genome as a template, primer pair P3 (SEQ ID N0:3) and P4 (SEQ ID NO: 4) to amplify the gene, BamHI and Xhol double digestion After loading pSJ3, pSJ3_feSecA is obtained.
  • Plasmid pSJ3-MSecA encodes (B. subtilis-H. influenzae) Chimeric SecA protein, MSecA, which is from amino acid position 1 to position 809 of Bacillus subtilis SecA protein and No. 867 to Haemophilus influenzae SecA protein.
  • the 901 amino acid was obtained by fusion.
  • the plasmid was constructed using the large primer PCR technique as follows: Using the Haemophilus influenzae genome as a template, primer pairs P5 (SEQ ID NO: 5) and P6 (SEQ ID NO: 6) amplification of Haemophilus influenzae SecA protein No. 867 The coding sequence for amino acid position 901. The fragment was recovered as a large primer, and matched with P3.
  • the coding sequence of MSecA protein was amplified by using plasmid pSJ3-feSe C A as a template. BamHI and Hindlll were double-digested and loaded into pSJ3 to obtain pSJ3-MSecA.
  • the plasmid pSJ3- ⁇ Se C A encodes (Bacillus subtilis-E. coli) chimeric SecA, ie Z ⁇ SecA protein, which is from amino acid position 1 to position 809 of Bacillus subtilis SecA protein and E. coli SecA protein
  • the amino acids from 870 to 901 are obtained by fusion. Using the E.
  • the primer pair P7 (SEQ ID NO: 7) and P2 amplify the coding sequence of amino acids 870 to 901 of the E. coli SecA protein.
  • the fragment was recovered as a large primer, and matched with P3, and the coding sequence of Z ⁇ SecA protein was amplified by using plasmid pSJ3-feSe C A as a template, and BamHI was digested and loaded into pSJ3 to obtain pSJ3- ⁇ Se C A .
  • Plasmid P SJ2-e>cSecB encodes E. coli SecB protein, using E. coli genome as template, primer pair P8 (SEQ ID NO: 8)
  • the sec gene was amplified with P9 (SEQ ID NO: 9), and BamHI and Hindi 11 were double-digested and loaded into pSJ2 to obtain pSJ2_e>cSecB.
  • the plasmid constructed in a) and b) was used to purify the corresponding protein in E. coli. These purified proteins were subsequently used in in vitro binding experiments and isothermal titration experiments.
  • the plasmid pMA5-e>cMalEll encodes a mutant of the E. coli maltose-binding protein (MalE), the MalEll protein, which has a three-amino acid substitution at the amino terminus (as shown in Figure 7A).
  • the mutation was introduced by the overlap extension PCR technique.
  • the plasmid construction process was as follows: Using the E.
  • the primer pair P10 (SEQ ID NO: 10) and Pll (SEQ ID NO: 11) amplified the ffla f gene signal peptide coding region
  • primer The ffla f gene mature peptide coding region was amplified for P12 (SEQ ID NO: 12) and P13 (SEQ ID NO: 13).
  • the primer pairs P10 and P13 were amplified to obtain the coding sequence of the full-length MalEll protein (mutations were introduced by P10 and P12, respectively), and Ndel and Hindlll were double-digested and loaded into pMA5.
  • the pMA5_e>cMalEll was obtained, and the plasmid diagram is shown in Fig. 2.
  • PhoA encodes E. coli alkaline phosphatase (PhoA), and its construction process is as follows: Escherichia coli genome as template, primer pair P14 (SEQ ID NO: 14) and P15 (SEQ ID NO: 15) Amplification/gene, Ndel and Hindlll were digested and loaded into pMA5 to obtain pMA5_ecPhoA
  • the plasmid pMA5-(e> C MalE43 ⁇ 4 0 A) encodes a fusion protein of maltose-binding protein and alkaline phosphatase (MalE-PhoA) derived from Escherichia coli, and the coding sequence of the fusion protein was constructed by overlap extension PCR.
  • the plasmid construction process was as follows: Primer pair P16 (SEQ ID NO: 16) and P17 (SEQ ID NO: 17) were used to amplify the gene containing no stop codon; primer pair P18 (SEQ ID NO: 18) And P15 amplify the coding sequence encoding the region of the mature peptide region of PhoA.
  • the primer pair P16 and P15 were amplified to obtain the coding sequence of the full-length MalE-PhoA protein, and Ndel and Hindlll were double-digested and loaded into pMA5 to obtain pMA5-(ecMalE43 ⁇ 4oA).
  • Plasmid pAXOl-ecSecB encodes the E. coli SecB protein, using the E. coli genome as a template, primer pair P19 (SEQ ID NO: 19) and P20 (SEQ ID NO: 20) The sec gene was amplified, and BamHI was digested and loaded into pAXO1 to obtain pAX01-ecSecB.
  • the plasmid diagram is shown in Fig. 3.
  • Plasmid pAX01-e> C Se C BL75Q encodes the SecB75 mutant of Escherichia coli SecB protein.
  • the mutation was introduced by overlap extension PCR.
  • the plasmid construction process was as follows: Escherichia coli genome as template, primer pair P19 and P21 (SEQ ID NO: 21) Amplification of the fragment upstream of the sec gene mutation site, primer pair P22 (SEQ ID NO: 22) and P20 amplify a fragment downstream of the sec? gene mutation site, and the mutation is introduced by P22.
  • primer pairs P19 and P20 were amplified to obtain the coding sequence of the full-length SecBL75Q protein, and BamHI was digested and ⁇ to obtain pAXOl-ecSecBL75Q.
  • P23 (SEQ ID N0: 23) and P24 (SEQ ID) NO: 24) was used for the construction of pAX01-ecSecBE77K and pAX01_e>cSecBL75Q&E77K, respectively, which encode the mutants SecBE77K and SecBL75Q/E77K of the E. coli SecB protein.
  • Plasmid pOE- ⁇ SecA encodes (B. subtilis-E. coli) chimeric SecA, Z ⁇ SecA protein, using plasmid pSJ3-Z1 ⁇ 2SecA as a template, primer pair P25 (SEQ ID NO: 25) and P26 (SEQ ID NO: 26)
  • the coding sequence of Z1 ⁇ 2SecA protein was amplified, and Kpnl and SacI I were digested and loaded into pOE to obtain pOE- ⁇ SecA.
  • the plasmid diagram is shown in Fig. 4.
  • the plasmid pOE- ⁇ SecA encodes the Bacillus subtilis SecA protein, using the B. subtilis genome as a template, primer pair P25 and P27 (SEQ ID NO: 27) amplified gene, Kpnl and SacI I double-digested and loaded into pOE to obtain pOE - feSecA.
  • LB medium was used, and appropriate antibiotics were added and cultured overnight at 37 ° C with shaking.
  • the antibiotic concentration was: ampicillin 100 ug/ml, kanamycin 100 ug/ml, chloramphenicol 5 ug/ml, erythromycin 5 ug/ml.
  • E. coli transformation was transformed using the mature calcium method, see Molecular Cloning, Third Edition.
  • B. subtilis uses a widely used inorganic salt natural competent method for plasmid transformation [31].
  • the carboxy terminus of the SecA protein determines the SecA-SecB specific interaction.
  • the ability of the SecB protein to bind to the SecB protein was gradually lost as the "zinc binding motif" of the carboxy terminus of the SecA protein accumulated during the evolution of "harmful mutations".
  • Replacing the carboxy-terminal "zinc-binding motif" of this SecA protein with the corresponding portion of the SecA protein capable of interacting with the SecB protein enables the ability to bind SecB protein to the SecA protein which is not capable of binding to the SecB protein, and Bacillus subtilis SecA is taken as an example to illustrate.
  • Figure 6A shows a sequence comparison of the SecA protein "zinc binding motif" from different biological sources.
  • the motif is highly conserved, the mutation is significant.
  • mutations may cause their SecA protein to lose its ability to bind to the SecB protein.
  • Figure 6B shows Haemophilus influenzae SecA (abbreviated as iSecA), Escherichia coli SecA (acSecA), Bacillus subtilis SecA (Bacillus) Sub til is Seek, abbreviated as feSecA) and two chimeric SecA, Bacillus subtilis-H. influenzae chimeric SecA (abbreviated as MSecA) and Bacillus subtilis-E. coli SecA (abbreviated as Z ⁇ SecA) Terminal amino acid sequence. The position indicated by the arrow indicates the position at which the chimerism occurs when the chimeric SecA is constructed.
  • Figure 6C shows the in vitro binding of different SecA to SecB.
  • ⁇ SecA can neither bind to Haemophilus influenzae SecB (AiSecB) nor bind Escherichia coli SecB (ecSecB), see lanes 1 and 2.
  • ecSecB Escherichia coli SecB
  • the chimeric SecA obtained by replacing the "zinc-binding motif" of the ⁇ SecA protein with the corresponding portion of iSecA or ecSecA, ie, MSecA and 6e>SecA confers at least a SecB protein homologous to its "zinc-binding motif".
  • Ability see lanes 3, 4 and 6. This result clearly indicates that the carboxy terminus of the SecA protein determines the SecA-SecB-specific interaction and can alter the binding properties of SecB by replacing the carboxy-terminal "zinc-binding motif" of the SecA protein.
  • MalEl l is a mutant of E. coli-derived MalE, the signal peptide N region of this mutant has only one net positive charge (the wild type is 3 net positive charges) and the mature peptide region immediately following the signal peptide One net positive charge (wild type net charge is 0), hence the name MalEl l, as shown in Fig. 7A, the secretion efficiency of the mutant in Bacillus subtilis is lower than that of the wild type, so in the present invention This mutant was employed to highlight the functionality of the SecB-mediated post-translational targeting pathway, ie, to enhance the efficiency of host secretion of foreign proteins.
  • Three plasmids encoding feSecA or beSeck or empty vector pOE, encoding ecSecB or empty vector pAXO l and pMA5 encoding MalEl l were simultaneously transformed into Bacillus subtilis, and 6 strains expressing different combinations of SecA, ecSecB and MalEl l were obtained. . These strains were cultured for 15 hours in LB medium supplemented with 0.5% xylose-induced ecSecB expression, and samples were analyzed. The results are shown in Figure 7B. Expression of ecSecB alone, feSecA or Z ⁇ SecA could not increase the secretion of MalEl by the host.
  • the present invention provides two "reverse" evidences further supporting the conclusion of the present invention that co-expression of ⁇ SecA and ecSecB can reconstitute a SecB-mediated post-translational targeting pathway in B. subtilis, the presence of which can be increased The efficiency with which the host secretes foreign proteins.
  • the SecB-mediated post-translational targeting pathway is versatile in increasing the efficiency of host secretion of foreign proteins.
  • the exogenous secretory proteins E. coli-derived alkaline phosphatase (PhoA) and maltose-binding protein-alkaline phosphatase, are fused.
  • the protein (MalE-PhoA) is illustrated as an example.
  • Figure 9A details the secretion of PhoA in different combinations of SecA and ecSecB.
  • the amount of PhoA secreted in the medium can be reflected by the activity of PhoA. In the case of expressing ecSecB alone, the secretion of PhoA did not increase (compared with No. 1 and No. 1). It is worth noting that whether you express ⁇ SecA alone or Z ⁇ SecA (compared with No. 3 and No. 5 and No. 1), the amount of PhoA secretion increases by about 30%, which is consistent with the literature report that SecA alone can also The targeted pathway recognizes the nascent secreted peptide chain.
  • the immunoblot shown in Figure 9B demonstrates that co-expression of Z ⁇ SecA and ecSecB increases the amount of PhoA secreted. Although its "apparent secretion efficiency" is already high, no large amount of PhoA precursor is detected in the cellular fraction (lane 2). The inventors speculate that in the case of co-expression of be k and ecSecB, the targeting efficiency of PhoA is greatly increased, and some PhoA precursors are prevented from being degraded in the cytoplasm, thus in the presence of SecB-mediated targeting pathways, PhoA The amount of secretion has increased dramatically.
  • Figure 9C shows the effect of coexpression of Z1 ⁇ 2SecA and ecSecB on the secretion efficiency of MalE_PhoA.
  • Western blot confirmed that the secretion efficiency of MalE_PhoA was greatly increased in the case of co-expression of SecA and ecSecB, and the secretion of MalE-PhoA in the medium was greatly increased, and accordingly the accumulation of precursors in the cytoplasm was greatly reduced (lane 3). Compare with 4 and 1).
  • the enzyme activity assay in the medium confirmed the results of the immunoblotting, that is, the secretion of MalE-PhoA increased by more than 70%.
  • the chimeric portion (the last 32 amino acids) of the chimeric ⁇ SecA protein used in Example 1 to Example 4 includes, in addition to the "zinc-binding motif" (the precise boundary of which has not been reported yet, but is known
  • the last 22 amino acids contain the "zinc binding motif", see literature [33]), and also include several amino acids upstream.
  • Theoretically chimeric (lengths less than 22 amino acids or greater than 32 amino acids) chimeric SecA can at least bind to its "zinc binding motif" as long as it includes a "zinc binding motif” that mediates interaction with SecB.
  • Figure 10A shows that feSecA-R3 replaces only the last 22 amino acids of the carboxy terminus of the ⁇ SecA protein with the corresponding portion of ecSecA compared to Z ⁇ SecA. The resulting sequence was replaced as shown in SEQ ID NO:31.
  • the present invention relates to the construction and application of a SecB-mediated post-translational targeting pathway.
  • Bacillus subtilis is widely used in industry to produce (endogenous) secreted proteins; 2) Exogenous secreted proteins (such as those derived from Gram-negative bacteria) due to the significant differences in signal peptide characteristics between different taxa And the signal peptide of eukaryotic secretory protein) is inefficient in Bacillus subtilis; 3) SecB-mediated post-translational targeting pathway can effectively assist SecA to recognize the inferior nascent secreted peptide chain; 4) Bacillus subtilis The SecB protein is deleted, so the present invention attempts to reconstruct a SecB-mediated post-translational targeting pathway in Bacillus subtilis, in order to increase the efficiency of secretion of exogenous secreted proteins by the bacterium, and further expand the application of the bacterium to industrially produce secreted proteins.
  • the present invention constructs Z1 ⁇ 2S ec A based on the structural basis of the SecA-SecB interaction. That is, the last 32 amino acid residues at the carboxy terminus of the ⁇ SecA protein are replaced with the corresponding portion of the ecSecA protein, which contains a "zinc binding motif" that mediates the SecA-SecB interaction, thereby obtaining the ability to efficiently bind ecSecB.
  • cytoplasmic proteins can be recognized by the bacterial secretion system after artificial addition of signal peptides (ie, artificially secreted proteins), which are then secreted into the culture medium [26, 34]. Since the constructed artificial secreted proteins are often not optimized by signal peptides, the signal peptide efficiency of such proteins in B. subtilis is often low. In theory, the SecB-mediated post-translational targeting pathways of the present invention are also capable of increasing the efficiency of host secretion of artificial secreted proteins. Therefore, the present invention shows a broad application prospect.
  • Collier, DN, Escherichia coli signal peptides direct inefficient secretion of an outer membrane protein (OmpA) and periplasmic proteins (maltose-binding protein, ribose-binding protein, and alkaline phosphatase) in Bacillus subtilis. J Bacteriol, 1994. 176( 10): p. 3013-20.
  • OmpA outer membrane protein
  • periplasmic proteins maltose-binding protein, ribose-binding protein, and alkaline phosphatase

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Abstract

La présente invention concerne une construction fonctionnelle d'une voie d'adressage post-traductionnel faisant intervenir SecB et l'utilisation de cette construction. En particulier, l'invention concerne un procédé de construction d'une protéine SecA chimère ; de coexpression de la protéine SecA chimère et de la protéine SecB dans une bactérie, augmentant ainsi l'efficacité de sécrétion des protéines d'une bactérie dans laquelle a été supprimé le gène secB ; un procédé de construction d'une voie d'adressage post-traductionnel faisant intervenir SecB dans la bactérie dans laquelle a été supprimé le gène secB ; et un procédé d'amélioration de la capacité de sécrétion de protéines de la bactérie dans laquelle a été supprimé le gène secB. De plus, l'invention concerne également une séquence d'acides aminés connexe, une séquence de nucléotides, un vecteur d'expression, et un système contenant la séquence d'acides aminés, la séquence de nucléotides et/ou le vecteur d'expression.
PCT/CN2011/085121 2010-12-31 2011-12-31 Construction fonctionnelle d'une voie d'adressage post-traductionnel faisant intervenir secb et utilisation de cette construction WO2012089164A1 (fr)

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CN1236011A (zh) * 1998-05-15 1999-11-24 中国科学院上海生物化学研究所 一种使用分子伴侣促进蛋白质分泌的方法
WO2008123622A1 (fr) * 2007-04-02 2008-10-16 Kao Corporation Microorganisme modifie

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CN1236011A (zh) * 1998-05-15 1999-11-24 中国科学院上海生物化学研究所 一种使用分子伴侣促进蛋白质分泌的方法
WO2008123622A1 (fr) * 2007-04-02 2008-10-16 Kao Corporation Microorganisme modifie

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Title
COLLIER, D. N.: "Expression of Escherichia coli SecB in Bacillus subtilis Facilitates Secretion of the SecB-Dependent Maltose-Binding Protein of E coli.", JOURNAL OF BACTERIOLOGY., vol. 176, no. 16, 31 August 1994 (1994-08-31), pages 4937 - 4940 *
DATABASE GENBANK 1 September 2010 (2010-09-01), Database accession no. BAI87157.1 *
DATABASE GENBANK 24 September 2008 (2008-09-24), Database accession no. 1tf2_A *
MCNICHOLAS, P. ET AL.: "SecA proteins of Bacillus subtilis and Escherichia coli possess homologous amino-terminal ATP-binding domains regulating integration into the plasma membrane.", JOURNAL OF BACTERIOLOGY., vol. 177, no. 24, 31 December 1995 (1995-12-31), pages 7231 - 7237 *
ZHOU, J. H. ET AL.: "The structural view of bacterial translocation-specific chaperone SecB: implications for function.", MOLECULAR MICROBIOLOGY., vol. 58, no. 2, 9 September 2005 (2005-09-09), pages 349 - 357 *

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