CN114702559A - Bacillus subtilis protein PgsA and application thereof in surface display system - Google Patents

Bacillus subtilis protein PgsA and application thereof in surface display system Download PDF

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CN114702559A
CN114702559A CN202210352096.2A CN202210352096A CN114702559A CN 114702559 A CN114702559 A CN 114702559A CN 202210352096 A CN202210352096 A CN 202210352096A CN 114702559 A CN114702559 A CN 114702559A
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protein
display system
surface display
bacillus subtilis
corynebacterium crenatum
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黄明珠
宋卓琳
陈雪岚
涂宗财
李金林
张露
彭斌
胡明明
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Jiangxi Normal University
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Abstract

The invention discloses a bacillus subtilis protein PgsA and application thereof in a surface display system. Bacillus subtilis proteinsPgsAThe amino acid sequence of (1) is shown as SEQ ID NO. 1, the expression amount of the overexpression vector pXMJ19 in the corynebacterium crenatum is high, and the overexpression vector can be used for constructing a surface display system of the corynebacterium crenatum with high display efficiency. Uses the coding gene of green fluorescent protein and the coding gene of bacillus subtilis cell membrane binding proteinpgsAExpression in cells after fusionMiddle, green fluorescent protein binding protein through membranePgsADisplayed on the surface of the cell.

Description

Bacillus subtilis protein PgsA and application thereof in surface display system
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a bacillus subtilis protein PgsA and application thereof as an anchoring protein in a corynebacterium crenatum surface display system.
Background
The microbial cell surface display technology utilizes molecular biology means to fuse and introduce exogenous target protein or polypeptide gene and anchoring protein gene into a host, in particular to a technology for immobilizing protein or polypeptide on the surface of a microbial cell through specific anchoring protein, and the displayed target protein can keep the original spatial conformation and biological activity. The microbial cell surface display has wide application prospects in the aspects of whole-cell catalysts, polypeptide separation, whole-cell adsorbents, protein library screening, vaccine and antibody production, bioremediation, biosensors and the like.
At present, host bacteria which are applied in a microorganism surface display system mostly comprise bacteriophage, saccharomyces cerevisiae, escherichia coli, bacillus subtilis and the like. The corynebacterium crenatum is a gram-positive bacterium separated from soil by scientists in China, is widely applied to production of compounds such as amino acid, organic acid, alcohol and the like, belongs to food-grade microorganisms, has the advantages of low extracellular protease activity and the like, and has very wide application prospect on the surface of microbial cells. Currently, there is little development of display systems for Corynebacterium crenatum. In order to increase the versatility of the cell surface display technology of Corynebacterium crenatum, it is necessary to develop new and effective anchoring proteins.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the bacillus subtilis protein PgsA.
Another object of the present invention is to provide a gene encoding the Bacillus subtilis protein PgsA.
Still another object of the present invention is to provide a corynebacterium crenatum cell surface display system.
The invention also aims to provide a construction method of the corynebacterium crenatum cell surface display system.
The purpose of the invention is realized by the following technical scheme:
a Bacillus subtilis protein PgsA has an amino acid sequence shown in SEQ ID NO. 1.
The bacillus subtilis protein consists of 380 amino acids and is about 42.7KDa in size.
The nucleotide sequence of the gene for coding the Bacillus subtilis protein PgsA is shown as SEQ ID NO. 2.
The bacillus subtilis protein PgsA is used as an anchoring protein in a surface display system of corynebacterium crenatum.
The surface display system is a corynebacterium crenatum cell surface display system, the bacillus subtilis protein PgsA can be used for constructing the corynebacterium crenatum cell surface display system, and the corynebacterium crenatum cell surface display system is formed by fixing a target protein on the surface of a corynebacterium crenatum cell by taking the bacillus subtilis protein PgsA as an anchoring protein.
A corynebacterium crenatum cell surface display system is formed by fixing target protein on the surface of corynebacterium crenatum cells by taking bacillus subtilis protein PgsA as anchoring protein.
The target protein is fluorescent protein or any protein.
The construction method of the corynebacterium crenatum cell surface display system comprises the following steps:
(1) carrying out bypass PCR on a gene for coding the bacillus subtilis protein PgsA and a target protein gene to obtain a fusion fragment;
(2) inserting the fusion gene fragment obtained in the step (1) into an expression vector to construct a vector for expressing cell surface display protein;
(3) transforming the carrier constructed in the step (2) into corynebacterium crenatum, and then screening positive transformants according to the screening markers on the expression carrier to obtain the corynebacterium crenatum cell surface display system.
The construction method of the corynebacterium crenatum cell surface display system specifically comprises the following steps:
(i) constructing a recombinant plasmid by a homologous recombination mode of a gene for coding the bacillus subtilis protein PgsA, a gene for a target protein and an expression vector;
(ii) and (e) transforming the recombinant plasmid obtained in the step (i) into corynebacterium crenatum, and picking positive clones to obtain the corynebacterium crenatum cell surface display system.
The gene sequence of the bacillus subtilis protein PgsA in the step (i) is shown as SEQ ID NO. 2.
The target protein in step (i) is enhanced green fluorescent protein (sfGFP) or other target protein.
The nucleotide sequence of the enhanced green fluorescent protein (sfGFP) is shown as SEQ ID NO: 3.
The expression vector in the step (i) is a conventional corynebacterium glutamicum/corynebacterium crenatum expression vector with chloramphenicol resistance; preferably, the Corynebacterium crenatum expression vector pXMJ 19.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention provides a bacillus subtilis protein PgsA which is a transmembrane protein of bacillus subtilis, and the verification proves that the fusion protein of the bacillus subtilis protein PgsA and the enhanced green fluorescent protein has very high expression quantity on the surface of corynebacterium crenatum, so that the fusion protein can be used for constructing a surface display system of corynebacterium crenatum with high display efficiency;
(2) the surface display system of the corynebacterium crenatum is formed by fixing target protein (such as fluorescent protein or other protein) on the surface of a corynebacterium crenatum cell by taking bacillus subtilis protein PgsA as anchoring protein, so that the expression efficiency of the endogenous anchoring protein of the surface display system of the corynebacterium crenatum is improved.
Drawings
FIG. 1 is a PCR ligation product by bridge of sfGFP gene, Bacillus subtilis protein PgsA gene, sfGFP gene and Bacillus subtilis protein PgsA gene.
FIG. 2 shows fluorescence microscopy images of a strain containing the recombinant plasmid pXMJ19-PgsA-sfGFP, a strain containing the recombinant plasmid pXMJ19-sfGFP and a negative control strain.
Detailed Description
The present invention will be described in detail with reference to embodiments, but the present invention is not limited thereto. Reagents, methods and apparatus according to the present invention are conventional in the art unless otherwise indicated. The following examples are given with no indication of particular experimental methods and conditions, and are generally in accordance with routine experimentation. Unless otherwise specified, the materials and reagents used in the present invention are commercially available.
The implementation case is as follows: surface display of sfGFP in Corynebacterium crenatum
(1) Cloning of the Bacillus subtilis protein PgsA Gene
According to the sequence of the Bacillus subtilis protein PgsA gene (SEQ ID NO:2), the target protein sfGFP gene and the sequence characteristics of pXMJ19, an amplification primer is designed:
P1：5'-ATTAATTAAGCTTGCATGCCTATGAAAAAAGAACTGAGCTTTCATG-3' (SEQ ID NO:4)；
P2：5'-TCCAGTGAAAAGTTCTTCTCGTTTATGCATTTTAGATTTTAGTTTATCGCTATGATCAA-3' (SEQ ID NO:5)。
the genome DNA of the bacillus subtilis is taken as a template, and P1 and P2 are taken as primers, and the gene sequence of the bacillus subtilis protein PgsA is amplified by a PCR method, wherein the amplification conditions are as follows: pre-denaturation at 95 ℃ for 5 minutes; another 30 cycles of: denaturation at 95 ℃ for 10 seconds, annealing at 60 ℃ for 10 seconds, and extension at 72 ℃ for 20 seconds; final extension at 72 ℃ for 10 min.
(2) Cloning of target protein sfGFP Gene
According to the sequence characteristics of the target protein sfGFP gene (SEQ ID NO:3), the Bacillus subtilis protein PgsA gene sequence (SEQ ID NO:2) and pXMJ19, amplification primers are designed:
P3：5'-TTGATCATAGCGATAAACTAAAATCTAAAATGCATAAACGAGAAGAACTTTTCACTGGA-3' (SEQ ID NO:6)；
P4：5'-CTGAATTCGAGCTCGGTACCCTTATTATTTGTAGAGCTCATCCATGCCATGT-3' (SEQ ID NO:7)。
the target protein sfGFP gene is amplified by a PCR method by taking pRSETB-SFGFP plasmid as a template and P3 and P4 as primers, wherein the amplification conditions are as follows: pre-denaturation at 95 ℃ for 5 minutes; another 30 cycles of: denaturation at 95 ℃ for 10 seconds, annealing at 60 ℃ for 10 seconds, and extension at 72 ℃ for 10 seconds; final extension at 72 ℃ for 10 min.
(3) Cloning of the vector pXMJ19 plasmid
Based on the sequence characteristics on plasmid pXMJ19, amplification primers were designed:
P5：5'-GGGTACCGAGCTCGAATTCAGCTTG-3'(SEQ ID NO:8)；
P6：5'-AGGCATGCAAGCTTAATTAATTCTGT-3'(SEQ ID NO:9)。
using pXMJ19 as a template and P5 and P6 as primers, and amplifying a sequence of a vector pXMJ19 by a PCR method, wherein the amplification conditions are as follows: pre-denaturation at 95 ℃ for 5 minutes; another 30 cycles of: denaturation at 95 ℃ for 10 seconds, annealing at 65 ℃ for 10 seconds, and extension at 72 ℃ for 1 minute; final extension at 72 ℃ for 5 min.
(4) Synthesis of PgsA-sfGFP Gene fusion fragment
And (2) taking the PCR product of the Bacillus subtilis protein PgsA gene obtained in the step (1) and the PCR product of the sfGFP green fluorescent protein gene obtained in the step (2) as templates, and taking P1 and P4 as primers, and amplifying a PgsA-sfGFP gene fusion fragment by a PCR method under the following amplification conditions: pre-denaturation at 95 ℃ for 5 minutes; another 30 cycles of: denaturation at 95 ℃ for 10 seconds, annealing at 62 ℃ for 10 seconds, and extension at 72 ℃ for 30 seconds; final extension at 72 ℃ for 5 min. And finally, agarose gel electrophoresis detection is carried out, and the size of an electrophoresis band is in accordance with expectation as shown in figure 1, so that the fusion result of the gene fragment is shown.
(5) Construction of vector pXMJ19-PgsA-sfGFP
The PCR product of the plasmid Pxmj19 obtained in step (3) and the PgsA-sfGFP gene fusion fragment obtained in step (1) were ligated by homologous recombination (using NovoRec one-step directed cloning kit), and the ligation system was transformed into E.coli host DH 5. alpha. purchased from Toluo harbor). And (3) screening transformants by using LB plates containing 25mg/L of chloramphenicol, extracting plasmids by using transformants with positive identification, identifying and sequencing, wherein the result shows that the sequence is correct.
(6) Construction and identification of recombinant Corynebacterium crenatum surface display system pXMJ19-PgasA-sfGFP
After the plasmid pXMJ19-PgsA-sfGFP obtained in step (5) was used to transform Corynebacterium crenatum by electroporation, positive transformants were picked by plating on LB plate containing chloramphenicol at 12.5 mg/L.
(7) Construction and identification of control Strain containing Pxmj19-sfGFP plasmid
Based on the sequence characteristics of the target protein sfGFP gene (SEQ ID NO:3) and pXMJ19, amplification primers were designed: p7: 5'-ATTAATTAAGCTTGCATGCCTATGCATAAACGAGAAGAACTTTT-3' (SEQ ID NO: 10).
Construction of a control strain containing pXMJ19-sfGFP plasmid: amplifying a gene sequence of sfGFP by a PCR method by taking sfGFP (SEQ ID NO:3) as a template and P5 and P4 as primers; amplifying a sequence of the plasmid pXMJ19 by a PCR method by taking the plasmid pXMJ19 as a template and P5 and P6 as primers; and then, carrying out homologous recombination and connection on the PCR product of the sfGFP gene and the PCR product of the sequence of the plasmid pXMJ19 by a one-step cloning method, and finally constructing a control strain containing the pXMJ19-sfGFP plasmid, wherein the specific preparation and identification methods are shown in the steps (1) - (6).
(8) Fluorescence microscopy analysis of recombinant Corynebacterium crenatum surface display System pXMJ19-PgasA-sfGFP
The recombinant strain containing pXMJ19-PgasA-sfGFP was inoculated into 25ml LB liquid medium containing 12.5mg/L and 0.5 mM IPTG (isopropyl. beta. -D-1-thiogalactoside), cultured at 30 ℃ and 220rpm for 8 hours, the sample was centrifuged and resuspended, and then it was smeared on a microscope slide, and finally observed by a fluorescence microscope, the recombinant strain containing pXMJ19-PgasA-sfGFP showed stronger surface fluorescence compared to the control strain (FIG. 2).
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> university of Master in Jiangxi
<120> Bacillus subtilis protein PgsA and application thereof in surface display system
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Met Lys Lys Glu Leu Ser Phe His Glu Lys Leu Leu Lys Leu Thr Lys
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Val Phe Val Leu Met Phe Ala Phe Met Trp Ala Gly Lys Ala Glu Thr
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Pro Lys Val Lys Thr Tyr Ser Asp Asp Val Leu Ser Ala Ser Phe Val
50 55 60
Gly Asp Ile Met Met Gly Arg Tyr Val Glu Arg Val Thr Glu Gln Lys
65 70 75 80
Gly Ala Ser Ser Ile Phe Gln Tyr Val Glu Pro Ile Phe Lys Ala Ser
85 90 95
Asp Tyr Val Ala Gly Asn Phe Glu Asn Pro Val Thr Tyr Lys Lys Asn
100 105 110
Tyr Glu Glu Ala Glu Lys Glu Ile His Leu Gln Thr Asn Lys Glu Ser
115 120 125
Val Lys Val Leu Lys Asp Met Asn Phe Thr Val Leu Asn Gly Ala Asn
130 135 140
Asn His Ala Met Asp Tyr Gly Ala Gln Gly Met Lys Asp Thr Leu Glu
145 150 155 160
Glu Phe Ser Lys His Asn Leu Asp Ile Val Gly Ala Gly Tyr Ser Leu
165 170 175
Ser Asp Ala Lys Lys Asn Ile Ser Tyr Gln Glu Val Asn Gly Val Thr
180 185 190
Ile Ala Thr Leu Gly Phe Thr Asp Val Ser Gly Lys Gly Phe Ala Ala
195 200 205
Lys Lys Asn Thr Pro Gly Val Leu Pro Ala Asp Pro Glu Ile Phe Ile
210 215 220
Pro Met Ile Ser Glu Ala Lys Lys His Ala Asp Val Val Val Val Gln
225 230 235 240
Ser His Trp Gly Gln Glu Tyr Asp Asn Asp Pro Asn Asp Arg Gln Arg
245 250 255
Gln Leu Ala Arg Ala Met Ser Asp Ala Gly Ala Asp Ile Ile Val Gly
260 265 270
His His Pro His Val Leu Glu Pro Ile Glu Val Tyr Asn Gly Thr Val
275 280 285
Ile Phe Tyr Ser Leu Gly Asn Phe Val Phe Asp Gln Gly Trp Thr Arg
290 295 300
Thr Arg Asp Ser Ala Leu Val Gln Tyr His Leu Lys Lys Asn Gly Thr
305 310 315 320
Gly His Phe Glu Val Thr Pro Ile Asp Ile His Glu Ala Thr Pro Ala
325 330 335
Pro Val Lys Lys Gly Ser Leu Lys Gln Lys Thr Ile Ile Arg Glu Leu
340 345 350
Thr Lys Asp Ser Asn Phe Ala Trp Glu Val Glu Asp Gly Lys Leu Thr
355 360 365
Phe Asp Val Asp His Ser Asp Lys Leu Lys Ser Lys
370 375 380
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atgaaaaaag aactgagctt tcatgaaaag ctgctgaagc tgacaaaaca gcaaaaaaag 60
aaaaccaata agcacgtatt tattgccatt ccgatcgttt ttgtcctcat gttcgctttt 120
atgtgggcgg gaaaagcaga aacgccgaag gtcaaaacat attctgatga cgttctctca 180
gcctcatttg tcggcgatat tatgatgggc cgctatgttg aaagagtcac ggaacaaaaa 240
ggggcaagca gcatttttca atacgttgag cctatcttta aagcatcaga ctacgtagcc 300
ggaaactttg aaaacccggt aacctataaa aagaactatg aggaagcaga aaaagaaatt 360
catctgcaga caaacaagga atcagtaaaa gtcctgaagg atatgaactt cacggtcctt 420
aacggcgcga ataaccacgc catggattac ggcgcacagg gcatgaagga tacacttgaa 480
gagttttcga aacataacct cgatatcgtt ggggctggat acagcttgag cgatgcgaaa 540
aagaatattt cataccaaga agtaaacggg gtaacaatcg cgacccttgg attcacagat 600
gtatccggca aaggtttcgc ggctaaaaag aatacaccgg gcgtgcttcc cgcagatccg 660
gaaatcttta tccctatgat ttcagaagcg aaaaaacatg cagatgtcgt tgtcgtacag 720
tcacactggg gacaagaata tgacaatgat ccaaacgacc gtcagcgtca gcttgcaaga 780
gctatgtctg atgcgggagc tgatatcatt gtcggccatc acccgcacgt tctagaaccg 840
attgaagtat ataacggaac cgtcattttc tacagcctcg gcaactttgt ttttgaccaa 900
ggctggacaa gaacgagaga cagtgcactg gttcagtatc acctgaagaa aaacggaaca 960
ggccactttg aagtgacgcc gattgatatc catgaagcga cgcctgcacc ggtgaaaaaa 1020
ggaagcctga aacaaaaaac gattattcgc gaactcacga aagattctaa tttcgcatgg 1080
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atgcataaac gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60
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aaactcaccc ttaaatttat ttgcactact ggaaaactac ctgttccgtg gccaacactt 180
gtcactactc tgacctatgg tgttcaatgc ttttcccgtt atccggatca catgaaacgg 240
catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaacgcac tatatctttc 300
aaagatgacg ggacctacaa gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt 360
aatcgtatcg agttaaaggg tattgatttt aaagaagatg gaaacattct tggacacaaa 420
ctcgagtaca actttaactc acacaatgta tacatcacgg cagacaaaca aaagaatgga 480
atcaaagcta acttcaaaat tcgccacaac gttgaagatg gttccgttca actagcagac 540
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ctgtcgacac aatctgtcct ttcgaaagat cccaacgaaa agcgtgacca catggtcctt 660
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<211> 46
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attaattaag cttgcatgcc tatgaaaaaa gaactgagct ttcatg 46
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<211> 59
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<213> Artificial Sequence (Artificial Sequence)
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ctgaattcga gctcggtacc cttattattt gtagagctca tccatgccat gt 52
<210> 8
<211> 25
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<213> Artificial Sequence (Artificial Sequence)
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gggtaccgag ctcgaattca gcttg 25
<210> 9
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
aggcatgcaa gcttaattaa ttctgt 26
<210> 10
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
attaattaag cttgcatgcc tatgcataaa cgagaagaac tttt 44

Claims (10)

1. A bacillus subtilis protein PgsA, characterized by: the amino acid sequence is shown as SEQ ID NO. 1.
2. A gene encoding the bacillus subtilis protein PgsA of claim 1, wherein: the nucleotide sequence is shown as SEQ ID NO. 2.
3. Use of the bacillus subtilis protein PgsA according to claim 1 as an anchor protein in a cell surface display system, wherein: the cell surface display system is a corynebacterium crenatum cell surface display system.
4. A Corynebacterium crenatum cell surface display system is characterized in that: the method is characterized in that the bacillus subtilis protein PgsA of claim 1 is used as an anchor protein, and a target protein is fixed on the surface of a corynebacterium crenatum cell.
5. The Corynebacterium crenatum cell surface display system of claim 4, wherein: the target protein is fluorescent protein.
6. The method for constructing a cell surface display system of Corynebacterium crenatum according to claim 4, comprising the steps of:
(1) obtaining a fusion fragment by using a gene encoding the bacillus subtilis protein PgsA as claimed in claim 1 and a target protein gene through bypass PCR;
(2) inserting the fusion gene fragment obtained in the step (1) into an expression vector to construct a vector for expressing cell surface display protein;
(3) transforming the carrier constructed in the step (2) into corynebacterium crenatum, and then screening positive transformants according to the screening markers on the expression carrier to obtain the corynebacterium crenatum cell surface display system.
7. The method for constructing a cell surface display system of Corynebacterium crenatum according to claim 6, comprising the steps of:
(i) constructing a recombinant plasmid by homologous recombination of a gene encoding the bacillus subtilis protein PgsA as claimed in claim 1, a gene encoding a target protein and an expression vector;
(ii) and (e) transforming the recombinant plasmid obtained in the step (i) into corynebacterium crenatum, and picking positive clones to obtain the corynebacterium crenatum cell surface display system.
8. The method for constructing a cell surface display system of Corynebacterium crenatum according to claim 7, wherein: the gene sequence encoding the Bacillus subtilis protein PgsA of claim 1 in step (i) is shown in SEQ ID NO. 2.
9. The method for constructing a cell surface display system of Corynebacterium crenatum according to claim 7, wherein the expression vector comprises: the target protein in step (i) is enhanced green fluorescent protein (sfGFP).
10. The method for constructing a cell surface display system of Corynebacterium crenatum according to claim 7, wherein: the expression vector in step (i) is a corynebacterium crenatum expression vector pXMJ 19.
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