CN114805505A - Surface protein ProH of corynebacterium crenatum and application of surface protein ProH in surface display system - Google Patents
Surface protein ProH of corynebacterium crenatum and application of surface protein ProH in surface display system Download PDFInfo
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Abstract
The invention discloses a corynebacterium crenatum surface protein ProH and application thereof in a surface display system. The amino acid sequence of the surface protein ProH of the corynebacterium crenatum is shown in SEQ ID NO 1, the expression amount of the protein ProH in the corynebacterium crenatum is high through an overexpression vector pXMJ19, and the protein can be used for constructing a surface display system of the corynebacterium crenatum with high display efficiency. Is a coding gene of green fluorescent protein and a coding gene of surface protein ProH of corynebacterium crenatumpgsAAfter fusion, the green fluorescent protein is expressed in the cell, and is displayed on the surface of the cell through the membrane-bound protein ProH.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a surface protein ProH of corynebacterium crenatum and application thereof as an anchoring protein in a surface display system of corynebacterium crenatum.
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 a surface protein ProH of corynebacterium crenatum.
Another object of the present invention is to provide a gene encoding the surface protein ProH of Corynebacterium crenatum.
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 surface protein ProH of corynebacterium crenatum has an amino acid sequence shown in SEQ ID NO. 1.
The corynebacterium crenatum protein consists of 56 amino acids and is about 6.02 KDa.
The nucleotide sequence of the gene for coding the surface protein ProH of the corynebacterium crenatum is shown as SEQ ID NO. 2.
The surface protein ProH of the corynebacterium crenatum is used as an anchoring protein in a surface display system of the corynebacterium crenatum.
The surface display system is a corynebacterium crenatum cell surface display system, the surface protein ProH of the corynebacterium crenatum can be used for constructing the surface display system of the corynebacterium crenatum cell, and the surface display system of the corynebacterium crenatum is formed by fixing a target protein on the surface of the corynebacterium crenatum cell by taking the surface protein ProH of the corynebacterium crenatum as an anchoring protein.
A Corynebacterium crenatum cell surface display system is formed by fixing target protein on the surface of a Corynebacterium crenatum cell by taking the surface protein ProH of the Corynebacterium crenatum 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 surface protein ProH of the corynebacterium crenatum 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 surface protein ProH of the corynebacterium crenatum, a gene for 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 for coding the surface protein ProH of the corynebacterium crenatum 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 surface protein ProH of corynebacterium crenatum, which is a transmembrane protein of corynebacterium crenatum, and verified that the fusion protein of the surface protein ProH of the corynebacterium crenatum and enhanced green fluorescent protein has very high expression quantity on the surface of the corynebacterium crenatum, so that the surface protein ProH can be used for constructing a surface display system of the 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 surface protein ProH of the corynebacterium crenatum 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 shows the products of PCR ligation of sfGFP gene, ProH gene of surface protein of Corynebacterium crenatum, sfGFP gene and ProH gene of surface protein of Corynebacterium crenatum.
FIG. 2 is a fluorescence microscope picture of a strain containing the recombinant plasmid pXMJ19-ProH-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 indicated, all 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 surface protein ProH gene of corynebacterium crenatum
According to the sequence of the surface protein ProH gene of the corynebacterium crenatum (SEQ ID NO:2), the target protein sfGFP gene and the sequence characteristics on pXMJ19, an amplification primer is designed:
P1：5'-ATTAATTAAGCTTGCATGCCTATGGATCTTTCCGTTCTCAAGGAA-3' (SEQ ID NO:4)；
P2：5'-CTCCTTTGCCGCTTCCTTGGCAGCCTCCTTTGCTGCTTCGGAAGAGAGCTTATCCAGGT-3' (SEQ ID NO:5)。
the genome DNA of the corynebacterium crenatum is taken as a template, P1 and P2 are taken as primers, and the surface protein ProH gene sequence of the corynebacterium crenatum is amplified by a PCR method under the following amplification conditions: pre-denaturation at 95 ℃ for 5 minutes; another 30 cycles of: denaturation at 95 ℃ for 5 seconds, annealing at 62 ℃ for 5 seconds, and extension at 72 ℃ for 5 seconds; final extension at 72 ℃ for 10 min.
(2) Cloning of target protein sfGFP Gene
According to the target protein sfGFP gene (SEQ ID NO:3), the surface protein ProH gene sequence of the corynebacterium crenatum (SEQ ID NO:2) and the sequence characteristics on pXMJ19, amplification primers are designed:
P3：5'-CCAAGGAAGCGGCAAAGGAGGCGGCCAAGGAGGCAGCAAAGATGCATAAACGAGAAGAA-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 ProH-sfGFP Gene fusion fragment
And (2) taking the PCR product of the surface protein ProH gene of the corynebacterium crenatum 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 ProH-sfGFP gene fusion fragment by a PCR method under the amplification conditions that: 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-ProH-sfGFP
The PCR product of the plasmid Pxmj19 obtained in step (3) and the fusion fragment of the ProH-sfGFP gene obtained in step (1) are subjected to homologous recombination (the step uses a NovoRec one-step directed cloning kit), and the ligation system is transformed into an Escherichia coli host DH5 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-ProH-sfGFP
After the plasmid pXMJ19-ProH-sfGFP obtained in step (5) was used to transform Corynebacterium crenatum by electrotransformation, positive transformants were picked up by plating on LB plates 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; then, homologous recombination and connection are carried out on the PCR product of the sfGFP gene and the PCR product of the sequence of the plasmid pXMJ19 through a one-step cloning method, and finally a control strain containing the pXMJ19-sfGFP plasmid is constructed, 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-ProH-sfGFP
The recombinant strain containing pXMJ19-ProH-sfGFP was inoculated into 25ml LB liquid medium containing 12.5mg/L and 0.5 mM IPTG (isopropyl. beta. -D-1-thiogalactopyranoside), cultured at 30 ℃ and 220rpm for 8 hours, the sample was centrifuged and resuspended, and then smeared on a microscope slide, and finally observed by a fluorescence microscope, and the recombinant strain containing pXMJ19-ProH-sfGFP showed stronger fluorescence than the surface of 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
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<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Asp Leu Ser Val Leu Lys Glu Thr Leu Gly Asn Tyr Glu Thr Phe
1 5 10 15
Gly Gly Asn Ile Gly Thr Ala Leu Lys Gly Ile Pro Thr Leu Leu Thr
20 25 30
Ser Ile Leu Asn Phe Phe Asp Asn Phe Gly Ala Leu Ala Asp Thr Thr
35 40 45
Gly Asn Asn Leu Asp Lys Leu Ser Ser
50 55
<210> 2
<211> 171
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atggatcttt ccgttctcaa ggaaaccctc ggcaactacg agaccttcgg tggcaacatc 60
ggaaccgctc tgaagggcat cccaaccctg ctcacttcca tccttaactt cttcgacaac 120
ttcggagctc tcgctgacac caccggcaac aacctggata agctctcttc c 171
<210> 3
<211> 720
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgcataaac gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60
gatgttaatg ggcacaaatt ttctgtccgt ggagagggtg aaggtgatgc tacaaacgga 120
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
cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 600
ctgtcgacac aatctgtcct ttcgaaagat cccaacgaaa agcgtgacca catggtcctt 660
cttgagtttg taactgctgc tgggattaca catggcatgg atgagctcta caaataataa 720
<210> 4
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
attaattaag cttgcatgcc tatggatctt tccgttctca aggaa 45
<210> 5
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ctcctttgcc gcttccttgg cagcctcctt tgctgcttcg gaagagagct tatccaggt 59
<210> 6
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ccaaggaagc ggcaaaggag gcggccaagg aggcagcaaa gatgcataaa cgagaagaa 59
<210> 7
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ctgaattcga gctcggtacc cttattattt gtagagctca tccatgccat gt 52
<210> 8
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
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 surface protein ProH of Corynebacterium crenatum, which is characterized in that: the amino acid sequence is shown as SEQ ID NO. 1.
2. A gene encoding the surface protein ProH of corynebacterium crenatum according to claim 1, characterized in that: the nucleotide sequence is shown as SEQ ID NO. 2.
3. Use of the surface protein proH of Corynebacterium crenatum according to claim 1 as an anchoring protein in a surface display system, characterized in that: the surface display system is a corynebacterium crenatum cell surface display system.
4. A Corynebacterium crenatum cell surface display system, which is characterized in that: the surface protein ProH of Corynebacterium crenatum of claim 1 as an anchor protein, wherein the target protein is immobilized on the cell surface of Corynebacterium crenatum.
5. The Corynebacterium crenatum cell surface display system of claim 4, wherein: the target protein is a 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 performing bypass PCR on a gene encoding the surface protein ProH of corynebacterium crenatum as claimed in claim 1 and a target protein gene;
(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) and (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 for coding a surface protein ProH of corynebacterium crenatum as claimed in claim 1, 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.
8. The method for constructing a cell surface display system of Corynebacterium crenatum according to claim 7, wherein: the gene sequence encoding the surface protein ProH of Corynebacterium crenatum described 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 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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| CN202210352090.5A CN114805505A (en) | 2022-04-02 | 2022-04-02 | Surface protein ProH of corynebacterium crenatum and application of surface protein ProH in surface display system |
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Citations (5)
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|---|---|---|---|---|
| CN101182516A (en) * | 2007-11-20 | 2008-05-21 | 华东理工大学 | Bacterium surface displaying novel system, method and applications |
| JP2010187586A (en) * | 2009-02-17 | 2010-09-02 | Kobe Univ | Method for presenting protein on cell surface layer of microorganism |
| CN102260331A (en) * | 2011-07-29 | 2011-11-30 | 华南理工大学 | Pichia pastoris wall protein Gcw34, surface display system constructed by same and construction method of surface display system |
| WO2014139862A1 (en) * | 2013-03-13 | 2014-09-18 | Autodisplay Biotech Gmbh | Improved surface display of functional proteins in a broad range of gram negative bacteria |
| CN113603756A (en) * | 2021-08-17 | 2021-11-05 | 华南理工大学 | Corynebacterium glutamicum membrane protein Ncgl2775, surface display system and construction method thereof |
-
2022
- 2022-04-02 CN CN202210352090.5A patent/CN114805505A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182516A (en) * | 2007-11-20 | 2008-05-21 | 华东理工大学 | Bacterium surface displaying novel system, method and applications |
| JP2010187586A (en) * | 2009-02-17 | 2010-09-02 | Kobe Univ | Method for presenting protein on cell surface layer of microorganism |
| CN102260331A (en) * | 2011-07-29 | 2011-11-30 | 华南理工大学 | Pichia pastoris wall protein Gcw34, surface display system constructed by same and construction method of surface display system |
| WO2014139862A1 (en) * | 2013-03-13 | 2014-09-18 | Autodisplay Biotech Gmbh | Improved surface display of functional proteins in a broad range of gram negative bacteria |
| CN113603756A (en) * | 2021-08-17 | 2021-11-05 | 华南理工大学 | Corynebacterium glutamicum membrane protein Ncgl2775, surface display system and construction method thereof |
Non-Patent Citations (3)
| Title |
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| NCBI: "WP_003853754.1，MULTISPECIES: PorH family porin [Corynebacterium]" * |
| TOSHIHIRO TATENO等: "Development of novel cell surface display in Corynebacterium glutamicum using porin" * |
| 赵越等: "吐温对钝齿棒杆菌产L-精氨酸代谢的影响" * |
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Application publication date: 20220729 |