CN107759675A - A kind of signal peptide and its application that secernment efficiency can be improved from bacillus subtilis - Google Patents

A kind of signal peptide and its application that secernment efficiency can be improved from bacillus subtilis Download PDF

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Publication number
CN107759675A
CN107759675A CN201710990081.8A CN201710990081A CN107759675A CN 107759675 A CN107759675 A CN 107759675A CN 201710990081 A CN201710990081 A CN 201710990081A CN 107759675 A CN107759675 A CN 107759675A
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signal peptide
bacillus subtilis
nucleotide sequence
seq
expressing
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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)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
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  • Microbiology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
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Abstract

The invention discloses a kind of signal peptide and its application that secernment efficiency can be improved from bacillus subtilis.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, the high expression of foreign gene can be realized, effective element is provided for bacillus subtilis expression alien gene, the expression of heat-resisting beta galactosidase and transglutaminase can be applied to.

Description

A kind of signal peptide and its application that secernment efficiency can be improved from bacillus subtilis
Technical field
It is more particularly to a kind of to improve secretion effect from bacillus subtilis the invention belongs to gene engineering technology field The signal peptide of rate and its application.
Background technology
Bacillus subtillis (Bacillus subttlis) is to be only second to Escherichia coli in prokaryotes research field The important model bacterium of (Escherichia coli), it is the comparatively ideal place of secreting, expressing foreign protein in current prokaryotic expression system It is main.Bacillus subtilis (B.subtilis) is a kind of Gram positive aerobic type bacillus, interior can give birth to degeneration-resistant spore, cell membrane Form it is simple in construction, without endotoxin；Only individual layer outer membrane, when secretory protein is by cell membrane, and added in cytoplasm After work folds, just it is directly ejected into nutrient solution；Genetic background is clear, has completed genome sequencing；Growth is fast, Yi Pei Support, higher bacterial concentration can be kept in large-scale production；The characteristics of molecule manipulation is simple, and it is appropriate for engineered strain The significant advantages such as transformation, have been widely used in the production of industrial enzyme.
In genetic engineering, signal peptide is the important component of bacillus subtilis expression excretory system.Signal peptide guides Whole polypeptide chain is combined with intracellular molecular chaperones or secretion signal recognition site, and then transmembrane transport is to extracellular.Derive from The albumen of gram-positive bacteria can typically utilize native signal peptide effective expression to secrete, albumen of its expression product to Host Strains Enzyme also has certain tolerance, but the secreting, expressing of heterologous protein is then more difficult, generally will be with bacillus extracellular protein Promoter and signal sequence fusion just can be such that secreting, expressing effectively carries out.Therefore, bacillus subtilis itself variety classes is studied High efficient expression and mechanism of secretion of the signal peptide for realizing foreign gene etc. it 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 one kind derives from bacillus subtilis Bacterium can improve the signal peptide of secernment efficiency.
Another object of the present invention is to provide the signal peptide that secernment efficiency can be improved from bacillus subtilis Encoding gene.
A further object of the present invention is to provide the signal peptide that can improve secernment efficiency from bacillus subtilis Application.
The purpose of the present invention is achieved through the following technical solutions：One kind can improve secernment efficiency from bacillus subtilis Signal peptide, its amino acid sequence is as shown in SEQ ID NO.1.
The gene of the coding signal peptide that secernment efficiency can be improved from bacillus subtilis, its nucleotides sequence column selection 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 restructuring table of gene containing any of the above-described signal peptide that secernment efficiency can be improved from bacillus subtilis Protection scope of the present invention is fallen within up to carrier, recombination engineering bacteria, transgenic cell line or expression cassette.
Described recombination engineering bacteria is that described can improve secernment efficiency from bacillus subtilis by any of the above-described Signal peptide Gene Fusion to the N-terminal for needing expressing gene, make the albumen of the gene code after fusion N-terminal fusion have it is corresponding Signal peptide；The genetic engineering bacterium that beta galactosidase ability improves preferably is secreted, or is the base that can secrete transglutaminase Because of engineering bacteria；More preferably Bacillus subtilis genes engineering bacteria.
The described signal peptide that secernment efficiency can be improved from bacillus subtilis is improving destination protein secreting, expressing In application.
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 SPyopL fragment electrophoretic figures in embodiment 2；Wherein, swimming lane M：marker DNA；Swimming lane 1 is SPyopL fragments.
Fig. 4 is the structure schematic diagram of expression plasmid pBEp43-SPyopL-proMTG in embodiment 2.
Fig. 5 is the SDS-PAGE that B.subtilis ATCC6051 (pBEp43-SPyopL-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-SPyopL-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-SpyopL：5′- GAGAGGAATGTCGACATGAAAAAACTTATTATGGCTTTAG-3′；R-SpyopL：5′- CGTTGTCCATCTCGAGTGCACTTATGTAAGAAGTGCCTAG-3 ') genomic DNA is expanded, 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 yopL genes, amino acid sequence after NCBI verifications For：MKKLIMALVILGALGTSYISA (SEQ ID NO.1), the nucleotides sequence for encoding the amino acid sequence is classified as： ATGAAAAAACTTATTATGGCTTTAGTTATCTTGGGCGCACTAGGCACTTCTTACATAAGTGCA(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-SPyopL-bgaB plasmids obtained using above-described embodiment 1 as template, primers F- SPyopL(5′-GAGAGGAATGTCGACATGAAAAAACTTATTATGGCTTTAG-3′)、R-SPyopL(5′- CGTTGTCCATCTCGAGTGCACTTATGTAAGAAGTGCCTAG-3 ') amplification about 100bp SPyopL 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 (SPyopL fragments) and plasmid (pBEp43-proMTG) connection, structure obtain MTG and express extracellular plasmid pBEp43- SPyopL-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-SPyopL-bgaB) and B.subtilis that will be obtained ATCC6051 (pBEp43-SPyopL-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 SPyopL secretions is shown Lactoside expression of enzymes, for enzyme activity in 30~48h expression highest, wherein 36h reaches highest enzyme activity 9.54U/mL.
(4)SDS-PAGE：B.subtilis ATCC6051 (pBEp43-SPyopL-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 SPyopL 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
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Met Lys Lys Leu Ile Met Ala Leu Val Ile Leu Gly Ala Leu Gly Thr
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Ser Tyr Ile Ser Ala
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atggagctcg gatccgaatt caagcttgtc gacctgcagt ctagactcga g 51
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Claims (8)

  1. A kind of 1. signal peptide that secernment efficiency can be improved from bacillus subtilis, it is characterised in that：Its amino acid sequence is such as Shown in SEQ ID NO.1.
  2. 2. the gene of the signal peptide that secernment efficiency can be improved from bacillus subtilis described in coding claim 1, its core 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 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. the restructuring of the gene containing the signal peptide that secernment efficiency can be improved from bacillus subtilis described in claim 2 Expression vector, recombination engineering bacteria, transgenic cell line or expression cassette.
  4. A kind of 4. recombination engineering bacteria, it is characterised in that：It will can be carried from bacillus subtilis described in claim 2 The Gene Fusion of the signal peptide of hypersecretion efficiency makes the N-terminal of the albumen of the gene code after fusion to the N-terminal for needing expressing gene Fusion has 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. the signal peptide that secernment efficiency can be improved from bacillus subtilis described in claim 1 is improving destination protein point Secrete the application in expression, it is characterised in that：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 bacillus subtilis Middle secreting, expressing.
CN201710990081.8A 2017-10-23 2017-10-23 A kind of signal peptide and its application that secernment efficiency can be improved from bacillus subtilis Pending CN107759675A (en)

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CN112708624A (en) * 2021-01-15 2021-04-27 山东大学 DNA fragment with promoter and coding signal peptide functions and application thereof in production of beta-mannase
CN112813065A (en) * 2021-01-15 2021-05-18 山东大学 DNA segment with promoter and coding signal peptide function and application thereof in production of rhamnogalacturonan lyase
CN112852808A (en) * 2021-01-15 2021-05-28 山东大学 DNA fragment with promoter and coding signal peptide functions and application thereof in production of alpha-L-arabinanase
CN112852807A (en) * 2021-01-15 2021-05-28 山东大学 DNA fragment with promoter and coding signal peptide functions and application thereof in producing alkaline pectinase
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Publication number Priority date Publication date Assignee Title
CN112708624A (en) * 2021-01-15 2021-04-27 山东大学 DNA fragment with promoter and coding signal peptide functions and application thereof in production of beta-mannase
CN112813065A (en) * 2021-01-15 2021-05-18 山东大学 DNA segment with promoter and coding signal peptide function and application thereof in production of rhamnogalacturonan lyase
CN112852808A (en) * 2021-01-15 2021-05-28 山东大学 DNA fragment with promoter and coding signal peptide functions and application thereof in production of alpha-L-arabinanase
CN112852807A (en) * 2021-01-15 2021-05-28 山东大学 DNA fragment with promoter and coding signal peptide functions and application thereof in producing alkaline pectinase
CN112852807B (en) * 2021-01-15 2022-05-03 山东大学 DNA fragment with promoter and coding signal peptide functions and application thereof in producing alkaline pectinase
CN112708624B (en) * 2021-01-15 2022-05-03 山东大学 DNA fragment with promoter and coding signal peptide functions and application thereof in production of beta-mannase
CN112852808B (en) * 2021-01-15 2022-05-03 山东大学 DNA fragment with promoter and coding signal peptide functions and application thereof in production of alpha-L-arabinanase
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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Application publication date: 20180306