CN107698667A - A kind of bacillus subtilis can be used for signal peptide and its application for improving secernment efficiency - Google Patents

A kind of bacillus subtilis can be used for signal peptide and its application for improving secernment efficiency Download PDF

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Publication number
CN107698667A
CN107698667A CN201710990058.9A CN201710990058A CN107698667A CN 107698667 A CN107698667 A CN 107698667A CN 201710990058 A CN201710990058 A CN 201710990058A CN 107698667 A CN107698667 A CN 107698667A
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signal peptide
bacillus subtilis
improving
seq
nucleotide sequence
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Inventor
潘力
刘欣
叶燕锐
王斌
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South China University of Technology SCUT
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South China University of Technology SCUT
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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
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/02—Aminoacyltransferases (2.3.2)
    • C12Y203/02013—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses signal peptide and its application that a kind of bacillus subtilis can be used for improving secernment efficiency.The amino acid sequence of the signal peptide is as shown in SEQ ID NO.1.The nucleotide sequence for encoding the signal peptide is selected from following any sequence：(a) nucleotide sequence or its complementary series as shown in SEQ ID NO.2；(b) carry out what one or more nucleotides substitution, missing or additions were obtained to the nucleotide sequence shown in SEQ ID NO.2, have and nucleotide sequence or its complementary series of the nucleotide sequence identical shown in SEQ ID NO.2 as signal peptide function.The invention provides a kind of signal peptide for improving secernment efficiency, and the secretion capacity of bacterial strain is remarkably improved after use, effective element is provided for bacillus subtilis expression alien gene.It is applied to the expression of heat-resisting beta galactosidase and transglutaminase in the present invention.

Description

A kind of bacillus subtilis can be used for signal peptide and its application for improving secernment efficiency
Technical field
The invention belongs to gene engineering technology field, more particularly to a kind of bacillus subtilis can be used for improving secernment efficiency Signal peptide and its application.
Background technology
Bacillus subtilis (Bacillus subtilis) is a kind of Gram positive aerobic type bacillus, can be interior raw degeneration-resistant Spore, the composition of cell membrane is simple in construction, without endotoxin；Only individual layer outer membrane, when secretory protein is by cell membrane, and thin After matrix is processed folding, just it is directly ejected into nutrient solution；Genetic background is clear, has completed genome sequencing, Understanding of the people to its genetic background and physiological property is only second to Escherichia coli (Escherichia coli)；Fermentation is simple, right Nutrition can keep higher bacterial concentration, therefore it has in the production of industrial enzyme without particular/special requirement in large-scale production Extensive use.
In genetic engineering, signal peptide is the important component for realizing heterologous protein secretion expression.Signal peptide is positioned at new One section of distinctive amino acid sequence of raw secretory protein and epicyte protein N-terminal, it has for the cross-film secernment efficiency of albumen Great influence.Signal peptide sequence does not have obvious conserved amino acid sequences typically, is typically tied by the N with positive charge Structure domain, the H structure domain rich in hydrophobic amino acid and the composition of the C-structure domain with negative electrical charge.Expressed in bacillus subtilis and be In system, the albumen from gram-positive bacteria can typically utilize native signal peptide effective expression to secrete, its expression product pair The protease of Host Strains also has certain tolerance, but the secreting, expressing of heterologous protein is then more difficult, generally will be with gemma Bacillus extracellular protein promoter and signal sequence fusion just can be such that secreting, expressing effectively carries out.Therefore, bacillus subtilis is studied High efficient expression and mechanism of secretion of itself different types of signal peptide for realizing foreign gene etc. is significant.
The content of the invention
The shortcomings that primary and foremost purpose of the present invention is to overcome prior art and deficiency, there is provided a kind of bacillus subtilis can use In the signal peptide for improving secernment efficiency.
The signal peptide for improving secernment efficiency another object of the present invention is to provide the bacillus subtilis can be used for Encoding gene.
A further object of the present invention is to provide the signal peptide that the bacillus subtilis can be used for raising secernment efficiency Using.
The purpose of the present invention is achieved through the following technical solutions：A kind of bacillus subtilis can be used for improving secernment efficiency Signal peptide, its amino acid sequence is as shown in SEQ ID NO.1.
The gene that the bacillus subtilis can be used for improving the signal peptide of secernment efficiency is encoded, its nucleotide sequence is selected from Following any sequence：
(a) nucleotide sequence or its complementary series as shown in SEQ ID NO.2；
(b) one or more nucleotides substitutions, missing or addition institute are carried out to the nucleotide sequence shown in SEQ ID NO.2 Obtain, have with the nucleotide sequence identical shown in SEQ ID NO.2 as the nucleotide sequence of signal peptide function or Its complementary series.
It can be used for the recombination expression of the gene for the signal peptide for improving secernment efficiency containing any of the above-described bacillus subtilis Carrier, recombination engineering bacteria, transgenic cell line or expression cassette fall within protection scope of the present invention.
Described recombination engineering bacteria is that can be used for any of the above-described bacillus subtilis to improve secernment efficiency The Gene Fusion of signal peptide makes the N-terminal fusion of the albumen of the gene code after fusion have corresponding letter to the N-terminal for needing expressing gene Number peptide；Preferably secrete the genetic engineering bacterium that beta galactosidase ability improves；Or it is the gene that can secrete transglutaminase Engineering bacteria；More preferably Bacillus subtilis genes engineering bacteria.
The signal peptide that described bacillus subtilis can be used for improving secernment efficiency is in destination protein secreting, expressing is improved Application.
Described destination protein is thermostable beta-galactosidase or transglutaminase.
Described protein secretion is expressed as the secreting, expressing in prokaryotic expression system；Divide preferably in bacillus subtilis Secrete expression.
Described bacillus subtilis is preferably bacillus subtilis ATCC6051 (B.subtilis ATCC6051).
The present invention is had the following advantages relative to prior art and effect：
1st, the present invention is by from bacillus subtilis homologous protein signal peptide storehouse, building various signal peptides and beta galactose The expression vector that glycosides enzyme coding gene is combined, with reference to high-throughout screening technique, beta galactosidase point can be improved by filtering out The signal peptide secreted.The expression vector that the signal peptide is combined with transglutaminase proenzyme encoding gene is built simultaneously, is realized The secreting, expressing of transglutaminase proenzyme.
2nd, the invention provides a kind of amino acid fragment, there is the signal peptide of very strong secreting, expressing activity, can realize outer The high expression of source gene, effective element is provided especially for bacillus subtilis expression alien gene.
3rd, the present invention can make the increase of beta galactosidase secretory volume by merging signal peptide, and enzyme activity is further carried It is high.
4th, the present invention realizes transglutaminase proenzyme secreting, expressing by merging signal peptide.
Brief description of the drawings
Fig. 1 is the PCR primer electrophoretogram that biobrick is expanded in embodiment 1；Wherein, swimming lane M is marker DNA, swimming lane 1 is biobrick pcr amplification product.
Fig. 2 is plasmid pBEp43s-biobrick-bgaB and plasmid pBEp43-SP-bgaB structures signal in embodiment 1 Figure.
Fig. 3 is amplification SPpgsE fragment electrophoretic figures in embodiment 2；Wherein, swimming lane M：marker DNA；Swimming lane 1 is SPpgsE fragments.
Fig. 4 is the structure schematic diagram of expression plasmid pBEp43-SPpgsE-proMTG in embodiment 2.
Fig. 5 is the SDS-PAGE that B.subtilis ATCC6051 (pBEp43-SPpgsE-proMTG) are expressed in embodiment 2 Running gel figure；Wherein, swimming lane M：marker；Swimming lane 1 is B.subtilis ATCC6051；Swimming lane 2 is B.subtilis ATCC6051(pBEp43-SPpgsE-proMTG)；Arrow represents destination protein MTG position.
Embodiment
With reference to embodiment, the present invention is described in further detail, but the implementation of the present invention is not limited to this.
Molecular biology experiment technology employed in following examples includes PCR amplifications, plasmid extraction, DNA fragmentation enzyme Cut, connect, gel electrophoresis etc. referring specifically to《Molecular Cloning:A Laboratory guide》(third edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Huang Peitangs etc. are translated, and 2002, Beijing:Science Press).
The screening of the signal peptide of the beta galactosidase efficient secretory expression of embodiment 1
With reference to the method for (Christian Degering et.Al, 2010), by from bacillus subtilis homologous protein In signal peptide storehouse, the expression vector that various signal peptides are combined with beta galactosidase encoding gene (bgaB) is built, with reference to height The screening technique of flux, the signal peptide for improving beta galactosidase secretion effect in various degree is obtained from clone, is specifically included Following steps：
(1) pBEp43-biobrick-bgaB carriers are built：With two sections of artificial synthesized fragment SEQ ID NO.3, SEQ ID The PCR fragment for the biobrick (biological building blocks) that NO.4 annealing extensions obtain carries restriction enzyme site Kpn I and Xho I, and use is restricted The PCR primer (such as Fig. 1) of endonuclease digestion and the 100bp sizes purified insertion plasmid pBE-P43-bgaB (presses patent document：Pan A kind of DNA fragmentations with promoter function of the such as power and application .CN201510074949.0 [P] .2015. are built) identical inscribe Enzyme position, obtain pBEp43s-biobrick-bgaB plasmids (such as Fig. 2), the biobrick of design PCR fragment such as SEQ ID Shown in NO.5.The PCR fragment base sequence of amplification is sequenced by Sanger to be confirmed.
(2) pBEp43-SP (signal peptide database)-bgaB carriers are built：Extract bacillus subtilis (Bacillus subttlis168, being purchased from Guangdong Province's Culture Collection) genomic DNA (Omega bacterial genomes DNA extraction agents box), use corresponding primer (F-SppgsE：5′- GAGAGGAATGTCGACATGAAATTTGTCAAAGCCATCTGGC-3′；R-SppgsE：5′- CGTTGTCCATCTCGAGAGCTGACATAAACAAGAACACGAT-3 ') choose signal peptide DNA expanded, by PCR primer return Receive (Omega DNA gels QIAquick Gel Extraction Kit), the above-mentioned pBEp43-biobrick-bgaB built is inserted by Cloning Transformation In carrier, the use of restriction enzyme site is Kpn I and Xho I, is built into pBEp43-SP-bgaB carriers (such as Fig. 2).
(3) the pBEp43-SP-bgaB carriers built are passed through into electricity going to Bacillus subtillis B.subtilis ATCC6051, specific method is with reference to non-patent literature record (Natalia P, Zakataeva, Oksana V et al.A simple method to introduce marker-free genetic modification into chromosome of naturally nontransformable Bacillus amyloliquefaciens strains[J].Appl Microbiol Biotechnol.2010,85:1201-1209), cloned in resistant panel (the μ g/mL of kanamycins 20) Son.
(4) picked clones uses 96 orifice plate cultures, by determining β-glucose galactose glycosides enzyme enzyme activity, it is determined that secretion is excellent The clone of change.The measure of β-glucose galactose glycosides enzyme enzyme activity：By 32 μ L nutrient solutions and 288 μ L 0.25%ONPG (o- Nitrophenyl- β-D-Galactopyranoside, ortho-nitrophenyl β-D- synthesis) mix, incubated at 55 DEG C 15min, reaction terminating add 320 μ L (w/w) Na2CO3.Reaction is in chromogenic reaction, and light absorption value is determined under 405nm wavelength.
(5) secretion optimization clone is sequenced, determines whether signal peptide is changed.Screen and obtain by above-mentioned steps The signal peptide of beta galactosidase secretion effect can be improved, is the signal peptide of pgsE genes, amino acid sequence after NCBI verifications For：MKFVKAIWPFVAVAIVFLFMSA (SEQ ID NO.1), the nucleotides sequence for encoding the amino acid sequence is classified as： ATGAAATTTGTCAAAGCCATCTGGCCG TTTGTTGCCGTAGCCATCGTGTTCTTGTTTATGTCAGCT(SEQ ID NO.2)。
The detection of the signal peptide expression of embodiment 2
(1) MTG (transglutaminase) extracellular expression plasmid is built：Patent document (is pressed with plasmid pBEp43-proMTG： The bacillus subtilis of the plant weight groups of the such as Pan Li mono- and its method .CN201210052578.2 [P] for producing transglutaminase .2012. build) be expression plasmid, the pBEp43-SPpgsE-bgaB plasmids obtained using above-described embodiment 1 as template, primers F- SPpgsE(5′-GAGAGGAATGTCGACATGAAATTTGTCAAAGCCATCTGGC-3′)、R-SPpgsE(5′- CGTTGTCCATCTCGAGAGCTGACATAAACAAGAACACGAT-3 ') amplification about 100bp SPpgsE fragments (see Fig. 3).With In-fusion methods (concrete operation method is shown in the HiFi DNA Assembly Master Mix of NEBuilder companies) will be believed Number peptide (SPpgsE fragments) and plasmid (pBEp43-proMTG) connection, structure obtain MTG and express extracellular plasmid pBEp43- SPpgsE-proMTG (see Fig. 4).First converted with chemical transformation to Escherichia coli (E.coli JM110), obtain positive colony Son, extracts plasmid after sequencing, and the method for electricity consumption conversion is converted to bacillus subtilis B.subtilis ATCC6051.
(2) the transformant B.subtilis ATCC6051 (pBEp43-SPpgsE-bgaB) and B.subtilis that will be obtained ATCC6051 (pBEp43-SPpgsE-proMTG) is incubated in 10mL LB culture mediums (the μ g/mL of kanamycins 20), 37 DEG C, 200rpm activates 12h, and the seed liquor of activation is inoculated in 50mL LB culture mediums into (the μ g/mL of kanamycins 20,1% (w/w) Portugal Grape sugar) inoculum concentration be 1% (volume ratio), 37 DEG C, 200rpm always fermented 48h, every sampling in 6 hours.
(3) measure of β-glucose galactose glycosides enzyme enzyme activity：As a result β-glucose half of signal peptide SPpgsE secretions is shown Lactoside expression of enzymes, for enzyme activity in 30~48h expression highest, wherein 36h reaches highest enzyme activity 6.64U/mL.
(4)SDS-PAGE：B.subtilis ATCC6051 (pBEp43-SPpgsE-proMTG) have been cultivated to 36h hair Zymotic fluid centrifuging and taking supernatant, supernatant run SDS-PAGE electrophoresis, make with wild-type B. subtilis B.subtilis ATCC6051 For control, protein adhesive figure, which is shown in 44-46KDa, that band and MTG albumen are in the same size, and wild type control is in this magnitude range Interior no band, experimental result illustrate that MTG pepsinogens are expressed (see Fig. 5).Illustrate that SPpgsE can be used as useful signal peptide to make For bacillus subtilis.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.
Sequence table
<110>South China Science ＆ Engineering University
<120>A kind of bacillus subtilis can be used for signal peptide and its application for improving secernment efficiency
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Signal peptide
<400> 1
Met Lys Phe Val Lys Ala Ile Trp Pro Phe Val Ala Val Ala Ile Val
1 5 10 15
Phe Leu Phe Met Ser Ala
20
<210> 2
<211> 66
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>The encoding gene of signal peptide
<400> 2
atgaaatttg tcaaagccat ctggccgttt gttgccgtag ccatcgtgtt cttgtttatg 60
tcagct 66
<210> 3
<211> 51
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ggtaccatgg cgttcagcaa catgtctgcg caggctgcgg ccggtgcaca t 51
<210> 4
<211> 51
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atggagctcg gatccgaatt caagcttgtc gacctgcagt ctagactcga g 51
<210> 5
<211> 102
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ggtaccatgg cgttcagcaa catgtctgcg caggctgcgg ccggtgcaca tatggagctc 60
ggatccgaat tcaagcttgt cgacctgcag tctagactcg ag 102
<210> 6
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> f-sppgse
<400> 6
gagaggaatg tcgacatgaa atttgtcaaa gccatctggc 40
<210> 7
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> r-sppgse
<400> 7
cgttgtccat ctcgagagct gacataaaca agaacacgat 40

Claims (9)

1. a kind of bacillus subtilis can be used for the signal peptide for improving secernment efficiency, it is characterised in that：Its amino acid sequence such as SEQ Shown in ID NO.1.
2. the gene that the bacillus subtilis described in claim 1 can be used for improving the signal peptide of secernment efficiency is encoded, its nucleosides Acid sequence is selected from following any sequence：
(a) nucleotide sequence or its complementary series as shown in SEQ ID NO.2；
(b) one or more nucleotides substitutions, missing or addition is carried out to the nucleotide sequence shown in SEQ ID NO.2 to be obtained , have and the nucleotide sequence identical shown in SEQ ID NO.2 is as the nucleotide sequence of signal peptide function or its is mutual Complementary series.
3. it can be used for the restructuring table of the gene for the signal peptide for improving secernment efficiency containing the bacillus subtilis described in claim 2 Up to carrier, recombination engineering bacteria, transgenic cell line or expression cassette.
A kind of 4. recombination engineering bacteria, it is characterised in that：Bacillus subtilis described in claim 2 can be used for improving The Gene Fusion of the signal peptide of secernment efficiency melts the N-terminal of the albumen of the gene code after fusion to the N-terminal for needing expressing gene Conjunction has corresponding signal peptide.
5. recombination engineering bacteria according to claim 4, it is characterised in that：Described recombination engineering bacteria is secretion The genetic engineering bacterium that beta galactosidase ability improves, or be the genetic engineering bacterium that can secrete transglutaminase.
6. the bacillus subtilis described in claim 1 can be used for the signal peptide for improving secernment efficiency improving destination protein secretion Application in expression, it is characterised in that：Described destination protein is thermostable beta-galactosidase or transglutaminase.
7. application according to claim 6, it is characterised in that：Described destination protein secreting, expressing is in prokaryotic expression system Secreting, expressing in system.
8. application according to claim 6, it is characterised in that：Described destination protein secreting, expressing is bacillus subtilis Middle secreting, expressing.
9. application according to claim 8, it is characterised in that：Described bacillus subtilis is bacillus subtilis ATCC6051。
CN201710990058.9A 2017-10-23 2017-10-23 A kind of bacillus subtilis can be used for signal peptide and its application for improving secernment efficiency Pending CN107698667A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114875013A (en) * 2022-06-21 2022-08-09 南京林业大学 Method for secreting natural intracellular beta-galactosidase by using recombinant bacillus subtilis

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120135498A1 (en) * 2009-08-03 2012-05-31 C-Lecta Gmbh Method for Producing Nucleases of a Gram Negative Bacterium While Using a Gram Positive Expression Host
CN103709236A (en) * 2013-12-23 2014-04-09 江南大学 Signal peptide capable of improving secretion efficiency, and applications thereof
CN106589067A (en) * 2016-12-21 2017-04-26 生工生物工程（上海）股份有限公司 Signal peptide and coding genes, protein secretory expression vector and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120135498A1 (en) * 2009-08-03 2012-05-31 C-Lecta Gmbh Method for Producing Nucleases of a Gram Negative Bacterium While Using a Gram Positive Expression Host
CN103709236A (en) * 2013-12-23 2014-04-09 江南大学 Signal peptide capable of improving secretion efficiency, and applications thereof
CN106589067A (en) * 2016-12-21 2017-04-26 生工生物工程（上海）股份有限公司 Signal peptide and coding genes, protein secretory expression vector and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GENBANK: AB910599.1: "Bacillus subtilis pgsE gene，complete cds", 《GENBANK》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114875013A (en) * 2022-06-21 2022-08-09 南京林业大学 Method for secreting natural intracellular beta-galactosidase by using recombinant bacillus subtilis

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