CN107698666A - A kind of signal peptide for effectively improving secretion and its application - Google Patents

A kind of signal peptide for effectively improving secretion and its application Download PDF

Info

Publication number
CN107698666A
CN107698666A CN201710990056.XA CN201710990056A CN107698666A CN 107698666 A CN107698666 A CN 107698666A CN 201710990056 A CN201710990056 A CN 201710990056A CN 107698666 A CN107698666 A CN 107698666A
Authority
CN
China
Prior art keywords
signal peptide
expressing
nucleotide sequence
seq
effectively improving
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201710990056.XA
Other languages
Chinese (zh)
Inventor
潘力
刘欣
叶燕锐
王斌
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
South China University of Technology SCUT
Original Assignee
South China University of Technology SCUT
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by South China University of Technology SCUT filed Critical South China University of Technology SCUT
Priority to CN201710990056.XA priority Critical patent/CN107698666A/en
Publication of CN107698666A publication Critical patent/CN107698666A/en
Pending legal-status Critical Current

Links

Classifications

    • 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

Landscapes

  • 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)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a kind of signal peptide for effectively improving secretion and its application.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 with very strong secreting, expressing activity, by merging signal peptide, the increase of beta galactosidase secretory volume can be made, transglutaminase proenzyme secreting, expressing can also be realized.

Description

A kind of signal peptide for effectively improving secretion and its application
Technical field
The invention belongs to gene engineering technology field, more particularly to a kind of signal peptide for effectively improving secretion and its should With.
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, therefore works as secretory protein by cell membrane, and After cytoplasm is processed folding, just it is directly ejected into nutrient solution；And it is less demanding to condition of culture, in scale Higher bacterial concentration can be kept in production, therefore it is in the production extensive application of industrial enzyme；People carry on the back to its heredity The understanding of scape and physiological property is only second to Escherichia coli (Escherichia coli).
In genetic engineering, signal peptide is the important component for realizing heterologous protein secretion expression.In bacillus subtilis In expression system, the albumen from gram-positive bacteria can typically utilize native signal peptide effective expression to secrete, and it is expressed Product also has certain tolerance to the protease of Host Strains, but the secreting, expressing of heterologous protein is then more difficult, generally will Being merged with bacillus extracellular protein promoter and signal sequence can just be such that secreting, expressing effectively carries out.Therefore, withered grass bud is studied High efficient expression and mechanism of secretion of the different types of signal peptide of spore bacillus itself for realizing foreign gene etc. is significant.
The content of the invention
Primary and foremost purpose the shortcomings that being to overcome prior art of the present invention and deficiency, there is provided a kind of to effectively improve secretion Signal peptide.
Another object of the present invention is to provide the encoding gene of the signal peptide that can effectively improve secretion.
A further object of the present invention is the application for providing the signal peptide that can effectively improve secretion.
The purpose of the present invention is achieved through the following technical solutions：A kind of signal peptide for effectively improving secretion, its amino acid Sequence is as shown in SEQ ID NO.1.
The gene of the signal peptide of secretion can be effectively improved described in coding, 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.
The recombinant expression carrier of gene containing any of the above-described signal peptide for effectively improving secretion, recombination engineering Bacterium, transgenic cell line or expression cassette fall within protection scope of the present invention.
Described recombination engineering bacteria is by the Gene Fusion of any of the above-described signal peptide for effectively improving secretion To the N-terminal for needing expressing gene, make the N-terminal fusion of the albumen of the gene code after fusion have corresponding signal peptide；Preferably secrete The genetic engineering bacterium that beta galactosidase ability improves；Or it is the genetic engineering bacterium that can secrete transglutaminase；More preferably Bacillus subtilis genes engineering bacteria.
Application of the described signal peptide for effectively improving secretion in destination protein secreting, expressing is improved.
Described destination protein is thermostable beta-galactosidase or transglutaminase.
Described destination protein secreting, expressing is the secreting, expressing in prokaryotic expression system；Preferably in bacillus subtilis Middle secreting, expressing.
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 SPcccA fragment electrophoretic figures in embodiment 2；Wherein, swimming lane M：marker DNA；Swimming lane 1 is SPcccA fragments.
Fig. 4 is the structure schematic diagram of expression plasmid pBEp43-SPcccA-proMTG in embodiment 2.
Fig. 5 is the SDS-PAGE that B.subtilis ATCC6051 (pBEp43-SPcccA-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-SPcccA-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-SpcccA：5′- GAGAGGAATGTCGACATGAAATGGAACCCGCTTATTCCAT-3′；R-SpcccA：5′- CGTTGTCCATCTCGAGTCCTTTTACTGATAAAAAGAAAGT-3 ' expands to genomic DNA, then by PCR primer Reclaim (Omega DNA gels QIAquick Gel Extraction Kit), the above-mentioned pBEp43-biobrick- built is inserted by Cloning Transformation In bgaB carriers, 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 cccA genes, amino acid sequence after NCBI verifications For：MKWNPLIPFLLIAVLGIGLTFFLSVKG (SEQ ID N are O.1), the nucleotides sequence for encoding the amino acid sequence is classified as： ATGAAATGGAACCCGCTTATTC CATTTTTGCTGATCGCTGTTTTAGGAATTGGTCTAACTTTCTTTTTATCAGTAAAAGGA(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-SPcccA-bgaB plasmids obtained using above-described embodiment 1 as template, primers F- SPcccA(5′-GAGAGGAATGTCGACATGAAATGGAACCCGCTTATTCCAT-3′)、R-SPcccA(5′- CGTTGTCCATCTCGAGTCCTTTTACTGATAAAAAGAAAGT-3 ') amplification about 110bp SPcccA 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 (SPcccA fragments) and plasmid (pBEp43-proMTG) connection, structure obtain MTG and express extracellular plasmid pBEp43- SPcccA-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-SPcccA-bgaB) and B.subtilis that will be obtained ATCC6051 (pBEp43-SPcccA-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 SPcccA secretions is shown Lactoside expression of enzymes, for enzyme activity in 30~48h expression highest, wherein 36h reaches highest enzyme activity 5.08U/mL.
(4)SDS-PAGE：B.subtilis ATCC6051 (pBEp43-SPcccA-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 SPcccA 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 signal peptide for effectively improving secretion and its application
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Signal peptide
<400> 1
Met Lys Trp Asn Pro Leu Ile Pro Phe Leu Leu Ile Ala Val Leu Gly
1 5 10 15
Ile Gly Leu Thr Phe Phe Leu Ser Val Lys Gly
20 25
<210> 2
<211> 81
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>The encoding gene of signal peptide
<400> 2
atgaaatgga acccgcttat tccatttttg ctgatcgctg ttttaggaat tggtctaact 60
ttctttttat cagtaaaagg a 81
<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-spccca
<400> 6
gagaggaatg tcgacatgaa atggaacccg cttattccat 40
<210> 7
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> r-spccca
<400> 7
cgttgtccat ctcgagtcct tttactgata aaaagaaagt 40

Claims (8)

  1. A kind of 1. signal peptide for effectively improving secretion, it is characterised in that：Its amino acid sequence is as shown in SEQ IDNO.1.
  2. 2. encoding the gene of the signal peptide for effectively improving secretion described in claim 1, its nucleotide sequence is selected from following appoint One 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. 3. recombinant expression carrier, the recombination of the gene containing the signal peptide for effectively improving secretion described in claim 2 Engineering bacteria, transgenic cell line or expression cassette.
  4. A kind of 4. recombination engineering bacteria, it is characterised in that：By the signal peptide for effectively improving secretion described in claim 2 Gene Fusion to the N-terminal for needing expressing gene, make the N-terminal fusion of the albumen of the gene code after fusion have corresponding signal peptide.
  5. 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. 6. application of the signal peptide for effectively improving secretion in destination protein secreting, expressing is improved described in claim 1, its It is characterised by：Described destination protein is thermostable beta-galactosidase or transglutaminase.
  7. 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. 8. application according to claim 6, it is characterised in that：Described destination protein secreting, expressing is in bacillus subtilis Secreting, expressing in bacterium.
CN201710990056.XA 2017-10-23 2017-10-23 A kind of signal peptide for effectively improving secretion and its application Pending CN107698666A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710990056.XA CN107698666A (en) 2017-10-23 2017-10-23 A kind of signal peptide for effectively improving secretion and its application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710990056.XA CN107698666A (en) 2017-10-23 2017-10-23 A kind of signal peptide for effectively improving secretion and its application

Publications (1)

Publication Number Publication Date
CN107698666A true CN107698666A (en) 2018-02-16

Family

ID=61181437

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710990056.XA Pending CN107698666A (en) 2017-10-23 2017-10-23 A kind of signal peptide for effectively improving secretion and its application

Country Status (1)

Country Link
CN (1) CN107698666A (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998029536A2 (en) * 1996-12-31 1998-07-09 Nexia Biotechnologies, Inc. Reversibly inactive synthetic beta-galactosidase
CN104031913A (en) * 2013-03-07 2014-09-10 华东理工大学 Expression apparatus used for secretory expression of exogenous proteins in Bacillus subtilis
CN105255925A (en) * 2015-11-03 2016-01-20 天津科技大学 Efficient preparation method and gene engineering bacteria of sucrose isomerase
CN105601720A (en) * 2016-03-11 2016-05-25 南京工业大学 Signal peptide capable of effectively improving protein secretion expression efficiency and application thereof
CN105861403A (en) * 2016-04-15 2016-08-17 中国农业科学院饲料研究所 Recombinant bacterium capable of achieving efficient secretion expression of soluble polysaccharide monooxygenase CBP21 and application of recombinant bacterium
CN106282221A (en) * 2016-08-16 2017-01-04 齐鲁工业大学 The construction method of a kind of secreting, expressing trehalose synthase gene engineering bacteria and application
CN107200772A (en) * 2017-05-10 2017-09-26 江西省科学院微生物研究所 A kind of signal peptide for optimizing keratinase Ker efficient secretory expressions and its application

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998029536A2 (en) * 1996-12-31 1998-07-09 Nexia Biotechnologies, Inc. Reversibly inactive synthetic beta-galactosidase
CN104031913A (en) * 2013-03-07 2014-09-10 华东理工大学 Expression apparatus used for secretory expression of exogenous proteins in Bacillus subtilis
CN105255925A (en) * 2015-11-03 2016-01-20 天津科技大学 Efficient preparation method and gene engineering bacteria of sucrose isomerase
CN105601720A (en) * 2016-03-11 2016-05-25 南京工业大学 Signal peptide capable of effectively improving protein secretion expression efficiency and application thereof
CN105861403A (en) * 2016-04-15 2016-08-17 中国农业科学院饲料研究所 Recombinant bacterium capable of achieving efficient secretion expression of soluble polysaccharide monooxygenase CBP21 and application of recombinant bacterium
CN106282221A (en) * 2016-08-16 2017-01-04 齐鲁工业大学 The construction method of a kind of secreting, expressing trehalose synthase gene engineering bacteria and application
CN107200772A (en) * 2017-05-10 2017-09-26 江西省科学院微生物研究所 A kind of signal peptide for optimizing keratinase Ker efficient secretory expressions and its application

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
XIN LIU 等: "Identification of strong promoters based on the transcriptome of Bacillus licheniformis", 《BIOTECHNOL LETT》 *
李志敏 等: "溶解性多糖单加氧酶CBP21的高效分泌表达及与几丁质酶的协同作用研究", 《中国农业科技导报》 *
陈晓月 等: "β-半乳糖苷酶在枯草芽孢杆菌中的分泌表达", 《中国生物工程杂志》 *

Similar Documents

Publication Publication Date Title
CN106754833B (en) Method for efficiently expressing pullulanase in bacillus subtilis and recombinant bacillus subtilis
CN107759675A (en) A kind of signal peptide and its application that secernment efficiency can be improved from bacillus subtilis
US7585674B2 (en) Host microorganisms
CN112501149B (en) Alkaline protease mutant and gene, engineering bacterium, preparation method and application thereof
CN105950640B (en) The kappa-carrageenan enzyme gene and recombinase preparation method obtained using marine bacteria as source
CN104611317B (en) A kind of method for improving L-ASP secreting, expressing
CN109321552A (en) A kind of novel Pullulanase and its gene, engineering bacteria and preparation method
CN104402975B (en) Anti-aging small peptide and preparation method thereof
CN107936096A (en) A kind of signal peptide that can effectively improve protein secretion efficiency and its application
CN108004239A (en) A kind of Novel promoter of high efficient expression protease
CN107674119A (en) A kind of bacillus subtilis can effectively improve signal peptide and its application of secretion
CN107200772A (en) A kind of signal peptide for optimizing keratinase Ker efficient secretory expressions and its application
EP2206788A1 (en) Recombinant microorganism
CN106701813A (en) Expression vector as well as construction method and application thereof
CN111808834B (en) Method for efficiently expressing high-temperature resistant alpha-amylase in bacillus subtilis, recombinant promoter and application
CN104031913B (en) A kind of expression equipment for being used for the secreting, expressing foreign protein in bacillus subtilis
CN105505931B (en) A kind of strong promoter and its application in raising Nattokinase expression
CN107602707A (en) The dcas9 ω fusion proteins of one species specificity regulation bacillus subtilis exogenous gene expression and its application
CN107698667A (en) A kind of bacillus subtilis can be used for signal peptide and its application for improving secernment efficiency
CN107698668A (en) A kind of bacillus subtilis can improve signal peptide and its application of secernment efficiency
CN107698666A (en) A kind of signal peptide for effectively improving secretion and its application
CN105018407A (en) Bacillus subtilis of secretory expression proline aminopeptidase and application thereof
CN112980753B (en) Glycoside hydrolase fusion expression system for secretion of exogenous proteins
CN110004101A (en) Method for constructing optimal Validase TSP Concentrate II loss of expression host for target protein amount body
CN107857802A (en) A kind of bacillus subtilis can improve signal peptide and its application of secernment efficiency

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20180216