CN107955814A - A kind of promoter for improving protein expression efficiency - Google Patents
A kind of promoter for improving protein expression efficiency Download PDFInfo
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- CN107955814A CN107955814A CN201711376849.9A CN201711376849A CN107955814A CN 107955814 A CN107955814 A CN 107955814A CN 201711376849 A CN201711376849 A CN 201711376849A CN 107955814 A CN107955814 A CN 107955814A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
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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)
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- 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)
- C12Y302/01056—Glucuronosyl-disulfoglucosamine glucuronidase (3.2.1.56), i.e. glycuronidase
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Abstract
The invention discloses a kind of promoter for improving protein expression efficiency, belong to promoter engineering field.Promoter P involved in the method for the inventionBH4It is in promoter PsrfAOn the basis of to its core space carry out mutation transformation and formed.In new promoter PBH4Under control transcription, during enhanced green fluorescent protein (GFP), P is usedBH4Expression quantity be to use PsrfA3.5 times, express glycuronidase A (GusA) when, use PBH4Enzyme activity is set to be improved from 0.6U/ml to 8.9 ± 0.1U/ml.
Description
Technical field
The present invention relates to a kind of promoter for improving protein expression efficiency, belong to promoter engineering field.
Background technology
Bacillus subtilis expression system is considered as GRAS grades of organism, functional protein can be secreted into culture medium
In, separation purifying technique is simple, is widely used today as the host strain of heterologous protein expression.Bacillus subtilis is applied the most
Extensive host strain is 168 bacterial strain of withered grass and the mutant strain (such as WB600, WB700, WB800 etc.) of the series of withered grass 168, this hair
Host strain selected to use is Bacillus subtilis 168 in bright.
Gene expression system is a complicated process, and withered grass expression system is not also very ripe at present.In recent years, scientific research
Worker obtains very big progress in terms of bacillus subtilis secretion expresses foreign protein, has established an effective external source egg
White withered grass expression system.And in the transformation process of carrier system, promoter regulation element plays a part of the most important thing, selects
Strong promoter can make clone gene high level expression.Therefore, from promoter, strong promoter element is designed, structure is ripe
Efficient withered grass expression system, is either all of great significance in theoretical research or actual production.
The content of the invention
The object of the present invention is to provide a kind of saltant type bacillus subtilis promoter and using the promoter in withered grass
The method of high efficient expression foreign protein in bacillus 168.In view of promoter is the critical elements of gene engineering expression carrier, it is right
Foreign gene expression levels have large effect, and the present invention is based on strong promoter gene order, using to promoter gene
Core element be oriented evolution mode obtain one raising vigor mutant promoters.The new startup of this method structure
Son effectively raises expression quantity of the heterologous protein in bacillus subtilis.
First purpose of the present invention is to provide a kind of promoter for improving protein secretion expression efficiency, contains SEQ ID
Nucleotide sequence shown in NO.1.
Second object of the present invention is to provide the carrier of the promoter containing the raising protein secretion expression efficiency.
Third object of the present invention is to provide the genetic engineering bacterium that a kind of protein expression efficiency improves, and is with containing described
Improve the carrier expressing protein of the promoter of protein secretion expression efficiency.
In one embodiment of the invention, the albumen includes but not limited to enzyme.
In one embodiment of the invention, the albumen include oxidoreducing enzyme, transferase, hydrolase, lyase,
Isomerase or synzyme.
Fourth object of the present invention is to provide the construction method of the genetic engineering bacterium, and the described method includes following step
Suddenly：(1) promoter that carrier has in itself is connected or replaced with carrier with the promoter described in claim 1, is obtained containing new
The plasmid of promoterNew promoter；(2) by plasmidNew promoterIt is connected with foreign gene, obtains the plasmid containing new promoterNew promoter-foreign gene；
(3) by plasmidNew promoter-foreign geneIt is transformed into bacterium or fungal cell.
The present invention the 5th purpose be to provide it is a kind of improve promoter secreting, expressing efficiency method, be to promoter-
The nucleotide sequence in 10 areas upstream carries out 3~7bp mutation.
In one embodiment of the invention, the mutation is that -10 area upstream 3bp bases carry out degeneracy mutation.
In one embodiment of the invention, the mutation is -10 area upstream 3bp bases progress degeneracy mutation, and
It has been mutated on the basis of 3bp bases and the 4bp bases of 3bp alkali yl upstreams has been mutated again.
In one embodiment of the invention, the method is using the promoter shown in SEQ ID NO.2 as parent's sequence
Row, p- 10th area upstream 3bp bases carry out degeneracy mutation, and adjacent to 3bp alkali yl upstreams on the basis of 3bp bases have been mutated
4bp bases be mutated again, obtain the mutant shown in SEQ ID NO.1.
In one embodiment of the invention, it is described to sport degeneracy mutation.
The promoter that the 6th purpose of the present invention is to provide the raising protein secretion expression efficiency contains albumen in production
Product in terms of application.
In one embodiment of the invention, the application includes preparing glycuronidase A.
In one embodiment of the invention, the application is that the gene that will encode glycuronidase A is described with containing
The carrier connection of the promoter of protein secretion expression efficiency is improved, converts and ferments into host cell.
In one embodiment of the invention, the gene of the coding glycuronidase A contains shown in SEQ ID NO.3
Nucleotide sequence.
In one embodiment of the invention, the host cell is bacillus subtilis.
In one embodiment of the invention, the host cell is bacillus subtilis 168.
In one embodiment of the invention, the fermentation is 8~52h of fermentation at 35~37 DEG C.
The Novel promoter, is PBH4.Obtained by initial start son structure of PsrfA.The plasmid vector,
It is pBBH4-GFP and pBBH4-gusA in the present invention.
Beneficial effects of the present invention：The present invention uses orthogenesis mode, and the mutation of foreign protein yield can be improved by obtaining
Body promoter PBH4, the promoter can in bacillus subtilis efficiently expressing exogenous gene.Mutant promoters PBH4Transcription is lived
Property is compared with original promoter PsrfAMore than 6 times, during enhanced green fluorescent protein (GFP) are improved, the expression quantity using PBH4 is to use
PsrfA3.5 times, express glycuronidase A (GusA) when, use PBH4Make maximum enzyme activity improve to 8.9 from 0.6U/ml ±
0.1U/ml。
Brief description of the drawings
Fig. 1：PsrfAPromoter mutation body storehouse construction strategy；
Fig. 2：Ability to express verification a of the different promoters in shaking flask level：Growth curve, b：GFP expresses curve, c：
24h fluorescence intensities compare, d：SDS-PAGE detects GFP expression quantity；Wherein WT is wild-type promoters PsrfAExpress GFP；BH4 is
Mutant promoter PBH4Express GFP；
Fig. 3：Expression of the GusA in recombined bacillus subtilis；a：Growth curve and enzyme activity determination, b：SDS-PAGE is examined
Survey；Wherein, BSGus is wild-type promoters PsrfAExpress GusA；BSBHGuS is mutant promoter PBH4Express GusA.
Embodiment
1st, the detection method of GFP fluorescence intensities：12000 × g of sample centrifuges 2min, collects thalline, and PBS buffer is washed 3 times,
Certain density thalline suspension is diluted to PBS, takes 200 μ L to 96 hole elisa Plates, is put into SynergyTM H4 fluorescence microplate readers
Detect fluorescence.Exciting light 495nm, absorbs light 525nm, detects fluorescence.
2nd, GusA Enzyme activity assays method：
In 96 hole elisa Plates, final concentration of 0.5mgmL is added into 50 μ L cell pyrolysis liquids-14-
Nitrophenyl β-d-glucuronide (PNPG) substrate solution (pH 7.0), 37 DEG C of reaction 5min.It is dense eventually to add 100 μ L
Spend the Na for 1M2CO3Terminate reaction.The light absorption value being put under SynergyTM H4 fluorescence microplate readers detection 405nm.Establishing criteria
Curve calculates enzyme activity.
Enzyme activity defines：It is per minute to be converted into needed for 1mmol product p-nitrophenols under conditions of pH is 7.0 at 37 DEG C
Enzyme amount is an enzyme-activity unit 1U.
3rd, culture medium：LB culture mediums (L-1)：Tryptone 10g, NaCl 10g, yeast extract 5g, pH 7.0, are prepared solid
Agar powder 20g is added during body culture medium.
4th, condition of culture：Recombinant strain is taken out from -80 DEG C of refrigerators, in the flat lining outs of the LB containing corresponding resistant, chooses
Take single bacterium colony 200rmin in the test tube containing 5mL LB culture mediums-1, 37 DEG C are incubated overnight.It is transferred to afterwards by 2% inoculum concentration
Cultivated in 250mL shaking flasks containing 50mL LB culture mediums.
5th, 168 method for transformation of bacillus subtilis：Choose single bacterium colony BS168 to be seeded in the SPI culture mediums of 2mL, 37 DEG C are shaken
Bed overnight incubation；100 μ L are taken from overnight culture, are seeded in 5mL SPI culture mediums, are started after 37 DEG C of shaking table culture 4-5h
Survey OD600.Work as OD600When about 1.0, pipette 200 μ L bacterium solutions and be forwarded in the SPII culture mediums of 2mL, in 37 DEG C, 100rmin-1
Shaking table is incubated 1.5h；Xiang Guanzhong adds 20 μ L l00 × EGTA (double (alpha-amido ethylether) tetraacethyls of ethylene glycol) solution, in 37
℃、100r·min-1500 μ L are dispensed per l.5mL centrifuge tube after 10min is cultivated in shaking table；Xiang Guanzhong is added by sequence verification just
True appropriate plasmid, pressure-vaccum mix and are positioned over 37 DEG C, 100rmin-1Shaking table in cultivate 2h；Culture terminates, and draws bacterium solution about
200 μ L uniformly apply corresponding selective tablet, and 37 DEG C are incubated overnight.
Embodiment 1：PsrfAPromoter mutation body storehouse construction strategy
With PsrfA-3bp- 1 and PsrfA-3bp- 2 (tables 1) are primer, with pBSG03 (be disclosed in C.Guan, W.Cui, J.Cheng,
L.Zhou,J.Guo,X.Hu,G.Xiao,Z.Zhou,Construction and development ofan auto-
regulatory gene expression system in Bacillus subtilis,Microb.Cell Fact.14
(2015)) it is template, to PsrfAPromoter (SEQID NO.1) σAIdentification core space " -10th area " upstream 3bp bases carry out degeneracy and dash forward
Become (Fig. 1).And with P on the basis of 3bp mutationsrfA-4bp- 1 and PsrfA-4bp- 2 (tables 1) are primer, the plasmid after being mutated with 3bp
For template, to PsrfAPromoter σAIdentify that core space " -10th area " upstream 4bp bases carry out degeneracy mutation, obtain PsrfAMutant,
It is named as PBH4。
1 primer of table
Embodiment 2：Promoter PBH4Shake flat experiment is verified
Obtained P will be screenedBH4Promoter is verified for shake flat experiment.By PBH4Promoter and carrier pBSG03 and coding are green
The GFP genes connection of color fluorescin, obtains pBBH4-GFP.PBBH4-GFP plasmids are converted to bacillus subtilis 168 and are felt
In by state cell, these transformants expression GFP activity is verified in 250ml flask systems, with the open country cultivated under the same conditions
Raw type bacterial strain is (with unmutated PsrfAPromoter expression is mutually homogenic, and carrier and host are consistent) as control.
The results show that to contain mutant promoter PBH4The growth curve of plasmid expression GFP and wild-type strain basic one
Cause (Fig. 2-a), and wild type and mutant promoter all cell log growth middle and later periods activity it is rapid improve (Fig. 2-
B), this meets PsrfAPromoter is activated this feature by colony induction signaling.Screen obtained mutant promoter PBH4Activity
It is substantially improved compared with wild-type promoters, after 24 hours, expression fluorescence intensity is wild-type promoters PsrfA3.5 times.SDS-
PAGE testing results are consistent with GFP fluoroscopic examination results (Fig. 2-d), to contain mutant promoter PBH4The table of plasmid expression GFP
It is significantly higher than wild-type promoters P up to amountsrfAExpression quantity.
Embodiment 3：Expression of the glycuronidase A in recombined bacillus subtilis
By mutant promoters P in embodiment 2BH4It is respectively used to glycuronidase A (GusA) (sequence such as SEQ ID NO.3 institutes
Show) expression, the gene GFP in initial carrier is mainly substituted for GusA, primer P by implementation processgusA- i1, PgusA- i2 is used
In amplification glycuronidase A genes, P from e. coli jm109 and genomegusA- v1, PgusA- v2 be used for from pBSG03 and
Amplification contains wild-type promoters PsrfA and mutant promoter P on pBBH4-GFP plasmidsBH4Carrier framework, final structure
Plasmid pBS-gusA and pBBH4-gusA.Two kinds of plasmids are converted into B. subtilis cell, culture, measure expression effect
Fruit.As shown in figure 3, after culture 8h, GusA expression quantity is improved to 3.31 ± 0.04U/ml from 0.17 ± 0.03U/ml；Cultivate 12h
Afterwards, GusA expression quantity is improved to 7.75 ± 0.04U/ml from 0.30 ± 0.04U/ml；After cultivating 28h, GusA expression quantity is from 0.43
± 0.03U/ml is improved to 8.9 ± 0.1U/ml.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various change and modification, therefore the protection model of the present invention
Enclosing be subject to what claims were defined.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>A kind of promoter for improving protein expression efficiency
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 607
<212> DNA
<213>Artificial sequence
<400> 1
atcgacaaaa atgtcatgaa agaatcgttg taagacgctc ttcgcaaggg tgtctttttt 60
tgcctttttt tcggtttttg cgcggtacac atagtcatgt aaagattgta aattgcattc 120
agcaataaaa aaagattgaa cgcagcagtt tggtttaaaa atttttattt ttctgtaaat 180
aatgtttagt ggaaatgatt gcggcatccc gcaaaaaata ttgctgtaaa taaactggaa 240
tctttcggca tcccgcatga aacttttcac ccatttttcg gtgataaaaa catttttttg 300
tggtaaactg aacggtagaa agataaaaaa tattgaaaac aatgaataaa tagccaaaat 360
tggtttctta ttagggtggg gtcttgcggt ctttatccgc ttatgttaaa cgccgcaatg 420
ctgactgacg gcagcctgct ttaatagcgg ccatctgttt tttgattgga agcactgctt 480
tttaagtgta gtactttggg ctatttcggc tgttagttca taagaattaa aagctgatat 540
ggataagaaa gagaaaatgc gttgcacatg ttcactgctt ataaagatta ggggaggtat 600
gacaatg 607
<210> 2
<211> 607
<212> DNA
<213>Artificial sequence
<400> 2
atcgacaaaa atgtcatgaa agaatcgttg taagacgctc ttcgcaaggg tgtctttttt 60
tgcctttttt tcggtttttg cgcggtacac atagtcatgt aaagattgta aattgcattc 120
agcaataaaa aaagattgaa cgcagcagtt tggtttaaaa atttttattt ttctgtaaat 180
aatgtttagt ggaaatgatt gcggcatccc gcaaaaaata ttgctgtaaa taaactggaa 240
tctttcggca tcccgcatga aacttttcac ccatttttcg gtgataaaaa catttttttc 300
atttaaactg aacggtagaa agataaaaaa tattgaaaac aatgaataaa tagccaaaat 360
tggtttctta ttagggtggg gtcttgcggt ctttatccgc ttatgttaaa cgccgcaatg 420
ctgactgacg gcagcctgct ttaatagcgg ccatctgttt tttgattgga agcactgctt 480
tttaagtgta gtactttggg ctatttcggc tgttagttca taagaattaa aagctgatat 540
ggataagaaa gagaaaatgc gttgcacatg ttcactgctt ataaagatta ggggaggtat 600
gacaatg 607
<210> 3
<211> 1806
<212> DNA
<213>Artificial sequence
<400> 3
ttacgtcctg tagaaacccc aacccgtgaa atcaaaaaac tcgacggcct gtgggcattc 60
agtctggatc gcgaaaactg tggaattgat cagcgttggt gggaaagcgc gttacaagaa 120
agccgggcaa ttgctgtgcc aggcagtttt aacgatcagt tcgccgatgc agatattcgt 180
aattatgcgg gcaacgtctg gtatcagcgc gaagtcttta taccgaaagg ttgggcaggc 240
cagcgtatcg tgctgcgttt cgatgcggtc actcattacg gcaaagtgtg ggtcaataat 300
caggaagtga tggagcatca gggcggctat acgccatttg aagccgatgt cacgccgtat 360
gttattgccg ggaaaagtgt acgtatcacc gtttgtgtga acaacgaact gaactggcag 420
actatcccgc cgggaatggt gattaccgac gaaaacggca agaaaaagca gtcttacttc 480
catgatttct ttaactatgc cgggatccat cgcagcgtaa tgctctacac cacgccgaac 540
acctgggtgg acgatatcac cgtggtgacg catgtcgcgc aagactgtaa ccacgcgtct 600
gttgactggc aggtggtggc caatggtgat gttagcgttg aactgcgtga tgcggatcaa 660
caggtggttg caactggaca aggcactagc gggactttgc aagtggtgaa tccgcacctc 720
tggcaaccgg gtgaaggtta tctctatgaa ctgtgcgtca cagccaaaag ccagacagag 780
tgtgatatct acccgcttcg cgtcggcatc cggtcagtgg cagtgaaggg cgaacagttc 840
ctgattaacc acaaaccgtt ctactttact ggctttggtc gtcatgaaga tgcggacttg 900
cgtggcaaag gattcgataa cgtgctgatg gtgcacgacc acgcattaat ggactggatt 960
ggggccaact cctaccgtac ctcgcattac ccttacgctg aagagatgct cgactgggca 1020
gatgaacatg gcatcgtggt gattgatgaa actgctgctg tcggctttaa cctctcttta 1080
ggcattggtt tcgaagcggg caacaagccg aaagaactgt acagcgaaga ggcagtcaac 1140
ggggaaactc agcaagcgca cttacaggcg attaaagagc tgatagcgcg tgacaaaaac 1200
cacccaagcg tggtgatgtg gagtattgcc aacgaaccgg atacccgtcc gcaaggtgca 1260
cgggaatatt tcgcgccact ggcggaagca acgcgtaaac tcgacccgac gcgtccgatc 1320
acctgcgtca atgtaatgtt ctgcgacgct cacaccgata ccatcagcga tctctttgat 1380
gtgctgtgcc tgaaccgtta ttacggatgg tatgtccaaa gcggcgattt ggaaacggca 1440
gagaaggtac tggaaaaaga acttctggcc tggcaggaga aactgcatca gccgattatc 1500
atcaccgaat acggcgtgga tacgttagcc gggctgcact caatgtacac cgacatgtgg 1560
agtgaagagt atcagtgtgc atggctggat atgtatcacc gcgtctttga tcgcgtcagc 1620
gccgtcgtcg gtgaacaggt atggaatttc gccgattttg cgacctcgca aggcatattg 1680
cgcgttggcg gtaacaagaa agggatcttc actcgcgacc gcaaaccgaa gtcggcggct 1740
tttctgctgc aaaaacgctg gactggcatg aacttcggtg aaaaaccgca gcagggaggc 1800
aaacaa 1806
Claims (10)
1. a kind of promoter, it is characterised in that contain the nucleotide sequence shown in SEQIDNO.1.
2. the carrier containing promoter described in claim 1.
A kind of 3. genetic engineering bacterium, it is characterised in that the carrier expressing protein described in claim 2.
4. genetic engineering bacterium according to claim 3, it is characterised in that the albumen includes enzyme.
5. the construction method of genetic engineering bacterium described in claim 3, it is characterised in that include the following steps：(1) with claim
Promoter described in 1 is connected or replaces the promoter that carrier has in itself with carrier, obtains the plasmid containing new promoterNew promoter；
(2) by plasmidNew promoterIt is connected with foreign gene, obtains the plasmid containing new promoterNew promoter-foreign gene；(3) by plasmidNew promoter-foreign gene
It is transformed into bacterium or fungal cell.
A kind of 6. method for improving promoter secreting, expressing efficiency, it is characterised in that be the nucleotide to the area of promoter -10 upstream
Sequence carries out 3~7bp mutation.
7. according to the method described in claim 6, it is characterized in that, 10 area upstream 3bp bases of the mutation Shi Dui- carry out degeneracy
Mutation；Or 10 area upstream 3bp bases of Xian Dui- carry out degeneracy mutation, and it has been mutated on the basis of 3bp bases to 3bp alkali yl upstreams
4bp bases be mutated again.
8. the method according to claim 6 or 7, it is characterised in that set out using the promoter shown in SEQIDNO.2 as parent
Sequence.
9. application of the promoter in terms of protein-contg product is produced described in claim 1.
10. application of the promoter in terms of fermenting and producing glycuronidase A described in claim 1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1240482A (en) * | 1996-11-18 | 2000-01-05 | 诺沃诺尔迪斯克生物技术有限公司 | Methods for producing polypeptides in surfaction mutants of bacillus cells |
| CN106916819A (en) * | 2017-04-28 | 2017-07-04 | 江南大学 | Bacillus subtilis promoter and its build and application that a kind of activity is improved |
-
2017
- 2017-12-19 CN CN201711376849.9A patent/CN107955814B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1240482A (en) * | 1996-11-18 | 2000-01-05 | 诺沃诺尔迪斯克生物技术有限公司 | Methods for producing polypeptides in surfaction mutants of bacillus cells |
| CN106916819A (en) * | 2017-04-28 | 2017-07-04 | 江南大学 | Bacillus subtilis promoter and its build and application that a kind of activity is improved |
Non-Patent Citations (3)
| Title |
|---|
| CHENG JINTAO等: "《Enhancement of a high efficient autoinducible expression system in Bacillus subtilis by promoter engineering》", 《PROTEIN EXPRESSION AND PURIFICATION》 * |
| HAN LAICHUANG等: "《Development of a novel strategy for robust synthetic bacterial promoters based on a stepwise evolution targeting the spacer region of the core promoter in Bacillus subtilis》", 《MICROBIAL CELL FACTORIES》 * |
| HAN LAICHUANG等: "《Fabrication and characterization of a robust and strong bacterial promoter from a semi-rationally engineered promoter library in Bacillus subtilis》", 《PROCESS BIOCHEMISTRY》 * |
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| CN107955814B (en) | 2020-02-07 |
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