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 PDFInfo
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- 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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- 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)
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- 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
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- 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
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- 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
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- 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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- Life Sciences & Earth Sciences (AREA)
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- Gastroenterology & Hepatology (AREA)
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- 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
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。
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Cited By (1)
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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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 |
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2017
- 2017-10-23 CN CN201710990058.9A patent/CN107698667A/en active Pending
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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)
Title |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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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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