CN113122557A - Expression vector of membrane protein AmtB and expression purification method thereof - Google Patents

Expression vector of membrane protein AmtB and expression purification method thereof Download PDF

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CN113122557A
CN113122557A CN202010048397.7A CN202010048397A CN113122557A CN 113122557 A CN113122557 A CN 113122557A CN 202010048397 A CN202010048397 A CN 202010048397A CN 113122557 A CN113122557 A CN 113122557A
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amtb
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membrane protein
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周敏
张苑桢
卢颖洪
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Nanjing University of Science and Technology
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Abstract

The invention discloses an expression vector of membrane protein AmtB and an expression purification method thereof. The nucleotide sequence of the expression vector is shown as SEQ ID NO.2, and the expression vector comprises: the bacteriophage T7 promoter; an Escherichia coli ribosome binding site which comprises an NcoI sequence CCATGG, an Escherichia coli membrane protein AmtB sequence shown in SEQ ID NO.1 and a BamHI site GGATCC; a tobacco etch virus cysteine protease cleavage site GAGAACCTGTACTTCCAATCC; NdeI restriction site CATATG; a hyper-folding Venus fluorescent protein coding sequence; XhoI cleavage site CTCGAG; 6 histidine sites and the stop codon TAG. The invention utilizes the fluorescent protein with rigid molecules as a screening marker and an expression process indicator, and the fluorescent protein can be folded rapidly due to the rigid structure at the molecular level, and can help the nitrogen-terminal membrane protein AmtB to stabilize the conformation thereof. The expression vector constructed by the invention can realize the mass expression of the membrane protein AmtB and can be used for the high-throughput screening of the subsequent novel antibiotics.

Description

Expression vector of membrane protein AmtB and expression purification method thereof
Technical Field
The invention belongs to the technical field of protein production, and relates to an expression vector of an ammonia channel protein (AmtB) and an expression and purification method thereof.
Background
The cell membrane is the boundary between the inside and the outside of the cell, is an important organelle, and plays important roles in information transfer, energy transmission and material exchange. The target of the drug action is often located on the cell membrane. It is currently known that more than 50% of the targets for drug action are membrane proteins. Therefore, the method has very important significance for the research of the membrane protein.
However, since the membrane protein has a few natural states and a multi-transmembrane structure, the membrane protein is easy to be folded mistakenly and has a complex structure, and thus, the large-scale preparation of the membrane protein is always a difficult point and a hot point. In China, no biological company can express transmembrane proteins for more than 12 times, and only one or two companies can express transmembrane proteins for four times.
Coli has been the first choice for protein expression as a commonly used expression host, however, even the proteins of e.coli themselves are difficult to express in large amounts in e.coli. Currently, abuse of antibiotics causes a dilemma that the antibiotics cannot resist drug-resistant bacteria within 50 years. Therefore, a great amount of expression membrane proteins are used as action targets of antibiotics, and a pilot technical support can be provided for high-throughput screening of subsequent non-denaturing mass spectra.
Disclosure of Invention
The invention aims to provide an expression vector for expressing a large amount of membrane protein ammonia channel protein (AmtB) and an expression and purification method thereof. The invention establishes an efficient fusion expression system aiming at the escherichia coli membrane protein AmtB, and can be used for the subsequent further protein non-denaturation real-time monitoring mass spectrum research.
The technical scheme for realizing the purpose of the invention is as follows:
the expression vector of the membrane protein AmtB takes the hyper-folding Venus fluorescent protein (Chinese patent application 201910347575.3) which is obtained in the laboratory and used for positioning the Chlamydomonas reinhardtii protein as an initial protein skeleton, and obtains a fluorescent protein expression label with rigid molecules by further optimizing the composition of codons used for sequence expression, and is suitable for large-scale expression of the Escherichia coli membrane protein. The invention utilizes the fluorescent protein with rigid molecules as a screening marker and an expression process indicator, and the fluorescent protein can be folded rapidly due to the rigid structure at the molecular level and helps the nitrogen-terminal membrane protein AmtB to stabilize the conformation.
The expression vector of the membrane protein AmtB is expression plasmid pLy077-AmtB (SEQ ID NO.2), and comprises: the bacteriophage T7 promoter; an Escherichia coli ribosome binding site comprising an NcoI sequence CCATGG, an Escherichia coli membrane protein AmtB sequence (SEQ ID NO.1) and a BamHI site GGATCC; a tobacco etch virus cysteine protease cleavage site GAGAACCTGTACTTCCAATCC; NdeI restriction site CATATG; a hyper-folding Venus fluorescent protein coding sequence; XhoI cleavage site CTCGAG; 6 histidine sites and the stop codon TAG.
The invention also provides an expression and purification method of the expression vector of the membrane protein AmtB, which comprises the following specific steps:
step 1, preparing an escherichia coli membrane protein AmtB expression vector mutant strain: transforming an expression vector of the membrane protein AmtB, namely expression plasmid pLy077-AmtB into host escherichia coli, coating the host escherichia coli on an agar plate containing antibiotics and 0.1mM IPTG, and selecting yellow-green positive monoclonal bacteria for amplification culture and induced expression;
and 2, inoculating the selected mutant strain transformant into a culture medium for amplification culture, and carrying out large-scale induction expression of the membrane protein AmtB by using 1mM IPTG.
Preferably, in step 1, the Escherichia coli is Escherichia coli BL21(DE3) or Escherichia coli C43(DE 3).
Preferably, in step 2, the culture medium is an LB culture medium or a TB culture medium.
The invention introduces the target gene and the codon optimized hyper-folding Venus fluorescent protein with molecular rigidity into the expression vector by fully synthesizing the expression vector, and greatly improves the transcription and translation efficiency of the target gene by optimizing the primary sequence of the nucleic acid coding. In addition, the folding speed of the carboxyl-terminal rigid fluorescent protein is controlled, so that the target membrane protein is helped to be correctly folded and inserted into the membrane. The rigid fluorescent protein can be used for monitoring the real-time expression process of the protein and protecting the carboxyl terminal of the protein, so that the target protein is enriched. The invention realizes high-flux screening of novel antibiotics through mass expression and later purification of escherichia coli membrane protein, and has important commercial prospect.
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FIG. 1 is a diagram showing the result of PCR agarose gel electrophoresis of the coding sequence of the AmtB protein.
FIG. 2 is a schematic diagram of the structure of the pLy077-AmtB plasmid.
FIG. 3 is a diagram showing the results of PCR verification of pLy077-AmtB colonies.
FIG. 4 is a diagram showing the results of single-restriction double-restriction agarose gel electrophoresis of NcoI and BamHI of plasmid pLy 077-AmtB.
FIG. 5 is a fluorescence image of colonies on transformed agar plates.
FIG. 6 is fluorescence imaging after induction and comparison of whole mycoprotein before and after induction.
FIG. 7 is a graph showing the expression results of BL21DE3 expression strain induced in TB medium for 6 hours.
Detailed Description
The invention will be further described with reference to specific embodiments and figures, but is not limited thereto. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
The materials used in the examples are as follows:
1. cell source
Coli strains DH5 α, BL21(DE3), C41(DE3), C43(DE3) were purchased from Wuhanling vast Bio Inc.
2. Source of plasmids
The pLy077 plasmid and the AmtB coding sequence were synthesized by Shanghai Biotechnology Ltd, the pLy077 plasmid containing: the bacteriophage T7 promoter; coli ribosome binding site comprising the NcoI sequence CCATGG and BamHI site GGATCC; a tobacco etch virus cysteine protease cleavage site GAGAACCTGTACTTCCAATCC; NdeI restriction site CATATG; a hyper-folding Venus fluorescent protein coding sequence; XhoI cleavage site CTCGAG; 6 histidine sites and the stop codon TAG. pLy077 plasmid and AmtB coding sequence are shown in the sequence table.
3. Source of primers
The synthetic primers were all from Biotechnology, Inc. of Ongbenaceae, Beijing.
4. Primary reagent
Tryptone, yeast extract, NaCl, Tris-baes were purchased from Sigma; restriction endonucleases, phusion enzymes, were purchased from Thermo Fisher; rTaq enzyme, T4 ligase, was purchased from Takara. The plasmid miniprep kit and the gel recovery kit were purchased from Axygen corporation. NTP, DEPC water and RNase inhibitor were purchased from Shanghai Biotechnology Ltd.
EXAMPLE 1 cloning of the AmtB Gene
Cloning of AmtB
a) A brand new AmtB coding sequence (SEQ ID NO.1) was designed and synthesized based on comparison of codon usage of the AmtB sequence with that of Escherichia coli codon in Kazusa online database (http:// www.kazusa.or.jp/codon /). And then amplified by a PCR method. The PCR reaction system configuration is shown in Table 1.
TABLE 1 PCR reaction System preparation Table
Figure BDA0002370232610000031
Figure BDA0002370232610000041
The PCR results are shown in FIG. 1.
b) Recovering target DNA, cutting the target fragment and pLy077 plasmid by using Nco I and BamHI enzyme, then carrying out agarose gel electrophoresis, and recovering a cut enzyme product; the recovered target fragment and the pLy077 plasmid fragment were added to a small centrifuge tube at a molar ratio of 5:1, and T4 ligase was added thereto and ligated at 16 ℃ overnight.
c) mu.L of the above ligation product was transformed into 80. mu.L of DH 5. alpha. competent cells by heat shock at 42 ℃ and 700. mu.L of LB medium was added thereto, followed by shaking at 37 ℃ and culturing at 200 rpm for 45 minutes.
d) Centrifuging the bacterial liquid at the rotating speed of 4000 rpm for 1 minute, and sucking 700 mu L of supernatant; after the remaining medium was gently aspirated by a pipette, the medium was spread on an LB solid plate containing ampicillin, and the plate was placed upside down in an incubator at 37 ℃ and cultured for 12 hours.
e) And (3) selecting a single colony in the plate, extracting a plasmid after a small amount of amplification, carrying out single enzyme digestion and double enzyme digestion on the extracted plasmid by using Nco I and BamHI, carrying out agarose gel electrophoresis identification, and carrying out sequencing identification to obtain an expression vector pLy077-AmtB of the membrane protein AmtB, wherein the structural schematic diagram of the plasmid is shown in FIG. 2. FIG. 3 is a diagram showing the results of PCR verification of pLy077-AmtB colonies. FIG. 4 is a diagram showing the results of single-restriction double-restriction agarose gel electrophoresis of NcoI and BamHI of plasmid pLy 077-AmtB.
Example 2 expression of AmtB
Inducible expression of fusion protein AmtB-superfolder fluorescent protein
The pLy077-AmtB plasmid which is identified correctly is transferred into an escherichia coli expression host Bl21(DE3) and C43(DE3) and is coated on a colony containing 0.1mM isopropyl thiogalactoside (IPTG) to obtain stable transformation; by detecting the fluorescence of the colonies, fig. 5 is a fluorescence map of the colonies on the transformed agar plate, and it can be seen from the presence or absence of fluorescence that whether the colonies contain the recombinant plasmid or not, and the colonies containing the recombinant plasmid show fluorescence, and it can be seen that a mutant strain expressing a large amount of the target protein is obtained.
Each colony was inoculated into LB and TB liquid media containing 100. mu.g/ml ampicillin, and cultured overnight at 37 ℃ at 200 rpm to give overnight-cultured bacteria. The overnight bacteria were inoculated into 750mL LB and TB medium at a ratio of 1:250 and cultured at 30 ℃ to OD at 230 rpm600Reaching 0.8-1.0, adding IPTG with final concentration of 0.5mM, and culturing at 25 deg.C for 2-6 hr. The cells were collected by centrifugation at 4000 rpm for 15 minutes. And (3) suspending the thalli in PBS buffer solution, and centrifuging for 15 minutes at 4000 rpm to obtain the thalli containing the AmtB fusion expression protein. FIG. 6 is fluorescence imaging after induction and comparison of whole mycoprotein before and after induction. As seen from FIG. 6, the expression level of CcmB protein was the highest after 6 hours of induction in TB medium using E.coli C43 as the expression strain. FIG. 7 shows BL21DE3 expression of the strains in TB medium for 6 hours.
Sequence listing
<110> Nanjing university of science and technology
Expression vector of <120> membrane protein AmtB and expression purification method thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1284
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgaaaattg cgaccattaa aaccggcctg gcgagcctgg cgatgctgcc gggcctggtg 60
atggcggcgc cggcggtggc ggataaagcg gataacgcgt ttatgatgat ttgcaccgcg 120
ctggtgctgt ttatgaccat tccgggcatt gcgctgtttt atggcggcct gattcgcggc 180
aaaaacgtgc tgagcatgct gacccaggtg accgtgacct ttgcgctggt gtgcattctg 240
tgggtggtgt atggctatag cctggcgttt ggcgaaggca acaacttttt tggcaacatt 300
aactggctga tgctgaaaaa cattgaactg accgcggtga tgggcagcat ttatcagtat 360
attcatgtgg cgtttcaggg cagctttgcg tgcattaccg tgggcctgat tgtgggcgcg 420
ctggcggaac gcattcgctt tagcgcggtg ctgatttttg tggtggtgtg gctgaccctg 480
agctatattc cgattgcgca tatggtgtgg ggcggcggcc tgctggcgag ccatggcgcg 540
ctggattttg cgggcggcac cgtggtgcat attaacgcgg cgattgcggg cctggtgggc 600
gcgtatctga ttggcaaacg cgtgggcttt ggcaaagaag cgtttaaacc gcataacctg 660
ccgatggtgt ttaccggcac cgcgattctg tatattggct ggtttggctt taacgcgggc 720
agcgcgggca ccgcgaacga aattgcggcg ctggcgtttg tgaacaccgt ggtggcgacc 780
gcggcggcga ttctgggctg gatttttggc gaatgggcgc tgcgcggcaa accgagcctg 840
ctgggcgcgt gcagcggcgc gattgcgggc ctggtgggcg tgaccccggc gtgcggctat 900
attggcgtgg gcggcgcgct gattattggc gtggtggcgg gcctggcggg cctgtggggc 960
gtgaccatgc tgaaacgcct gctgcgcgtg gatgatccgt gcgatgtgtt tggcgtgcat 1020
ggcgtgtgcg gcattgtggg ctgcattatg accggcattt ttgcggcgag cagcctgggc 1080
ggcgtgggct ttgcggaagg cgtgaccatg ggccatcagc tgctggtgca gctggaaagc 1140
attgcgatta ccattgtgtg gagcggcgtg gtggcgttta ttggctataa actggcggat 1200
ctgaccgtgg gcctgcgcgt gccggaagaa caggaacgcg aaggcctgga tgtgaacagc 1260
catggcgaaa acgcgtataa cgcg 1284
<210> 2
<211> 7279
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600
tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660
actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720
gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780
aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840
agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900
cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960
aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020
tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080
tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140
taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200
ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260
tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320
tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380
cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280
tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340
caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400
ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520
gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580
gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640
aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700
ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760
acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820
ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880
tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940
tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000
cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060
gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120
ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180
catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240
ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300
gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360
gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420
ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480
atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600
tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660
ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720
aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780
atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840
cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900
gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960
tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020
agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080
gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140
ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200
catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260
tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320
tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380
gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440
ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500
tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560
catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620
cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680
tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740
ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800
ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860
cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920
gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980
aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040
ttttgtttaa ctttaagaag gagatatacc atgggcatga aaattgcgac cattaaaacc 5100
ggcctggcga gcctggcgat gctgccgggc ctggtgatgg cggcgccggc ggtggcggat 5160
aaagcggata acgcgtttat gatgatttgc accgcgctgg tgctgtttat gaccattccg 5220
ggcattgcgc tgttttatgg cggcctgatt cgcggcaaaa acgtgctgag catgctgacc 5280
caggtgaccg tgacctttgc gctggtgtgc attctgtggg tggtgtatgg ctatagcctg 5340
gcgtttggcg aaggcaacaa cttttttggc aacattaact ggctgatgct gaaaaacatt 5400
gaactgaccg cggtgatggg cagcatttat cagtatattc atgtggcgtt tcagggcagc 5460
tttgcgtgca ttaccgtggg cctgattgtg ggcgcgctgg cggaacgcat tcgctttagc 5520
gcggtgctga tttttgtggt ggtgtggctg accctgagct atattccgat tgcgcatatg 5580
gtgtggggcg gcggcctgct ggcgagccat ggcgcgctgg attttgcggg cggcaccgtg 5640
gtgcatatta acgcggcgat tgcgggcctg gtgggcgcgt atctgattgg caaacgcgtg 5700
ggctttggca aagaagcgtt taaaccgcat aacctgccga tggtgtttac cggcaccgcg 5760
attctgtata ttggctggtt tggctttaac gcgggcagcg cgggcaccgc gaacgaaatt 5820
gcggcgctgg cgtttgtgaa caccgtggtg gcgaccgcgg cggcgattct gggctggatt 5880
tttggcgaat gggcgctgcg cggcaaaccg agcctgctgg gcgcgtgcag cggcgcgatt 5940
gcgggcctgg tgggcgtgac cccggcgtgc ggctatattg gcgtgggcgg cgcgctgatt 6000
attggcgtgg tggcgggcct ggcgggcctg tggggcgtga ccatgctgaa acgcctgctg 6060
cgcgtggatg atccgtgcga tgtgtttggc gtgcatggcg tgtgcggcat tgtgggctgc 6120
attatgaccg gcatttttgc ggcgagcagc ctgggcggcg tgggctttgc ggaaggcgtg 6180
accatgggcc atcagctgct ggtgcagctg gaaagcattg cgattaccat tgtgtggagc 6240
ggcgtggtgg cgtttattgg ctataaactg gcggatctga ccgtgggcct gcgcgtgccg 6300
gaagaacagg aacgcgaagg cctggatgtg aacagccatg gcgaaaacgc gtataacgcg 6360
ggatccggac tgcaggagaa cctgtacttc caatcccacc atatgtctaa aggtgaagaa 6420
ctgttcaccg gtgttgttcc gatcctggtt gaactggacg gtgacgttaa cggtcacaaa 6480
ttctctgttc gtggtgaagg tgaaggtgac gctaccaacg gtaaactgac cctgaaattc 6540
atctgcacca ccggtaaact gccggttccg tggccgaccc tggttaccac cctgacctac 6600
ggtgttcagt gcttctctcg ttacccggac cacatgaaac gtcacgactt cttcaaatct 6660
gctatgccgg aaggttacgt tcaggaacgt accatctctt tcaaagacga cggtacctac 6720
aaaacccgtg ctgaagttaa attcgaaggt gacaccctgg ttaaccgtat cgaactgaaa 6780
ggtatcgact tcaaagaaga cggtaacatc ctgggtcaca aactggaata caacttcaac 6840
tctcacaacg tttacatcac cgctgacaaa cagaaaaacg gtatcaaagc taacttcaaa 6900
atccgtcaca acgttgaaga cggttctgtt cagctggctg accactacca gcagaacacc 6960
ccgatcggtg acggtccggt tctgctgccg gacaaccact acctgtctac ccagtctgtt 7020
ctgtctaaag acccgaacga aaaacgtgac cacatggttc tgctggaatt cgttaccgct 7080
gctggtatca cccacggtat ggacgaactg tacaaactcg agcaccacca ccaccaccac 7140
tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag 7200
caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa 7260
ggaggaacta tatccggat 7279

Claims (4)

1. The expression vector of the membrane protein AmtB is expression plasmid pLy077-AmtB, and is characterized in that the nucleotide sequence is shown as SEQ ID NO.2 and comprises: the bacteriophage T7 promoter; an Escherichia coli ribosome binding site which comprises an NcoI sequence CCATGG, an Escherichia coli membrane protein AmtB sequence shown in SEQ ID NO.1 and a BamHI site GGATCC; a tobacco etch virus cysteine protease cleavage site GAGAACCTGTACTTCCAATCC; NdeI restriction site CATATG; a hyper-folding Venus fluorescent protein coding sequence; XhoI cleavage site CTCGAG; 6 histidine sites and the stop codon TAG.
2. The method for purifying the expression vector of the membrane protein AmtB according to claim 1, which comprises the following steps:
step 1, preparing an escherichia coli membrane protein AmtB expression vector mutant strain: transforming an expression vector of the membrane protein AmtB, namely expression plasmid pLy077-AmtB into host escherichia coli, coating the host escherichia coli on an agar plate containing antibiotics and 0.1mM IPTG, and selecting yellow-green positive monoclonal bacteria for amplification culture and induced expression;
and 2, inoculating the selected mutant strain transformant into a culture medium for amplification culture, and carrying out large-scale induction expression of the membrane protein AmtB by using 1mM IPTG.
3. The method for expression purification according to claim 2, wherein in step 1, the Escherichia coli is Escherichia coli BL21(DE3) or Escherichia coli C43(DE 3).
4. The method for purifying expression according to claim 2, wherein in the step 2, the culture medium is LB culture medium or TB culture medium.
CN202010048397.7A 2020-01-16 2020-01-16 Expression vector of membrane protein AmtB and expression purification method thereof Pending CN113122557A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104450593A (en) * 2013-09-25 2015-03-25 北京大学 Controllable escherichia coli acid-resistant system based on ion channel and application thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104450593A (en) * 2013-09-25 2015-03-25 北京大学 Controllable escherichia coli acid-resistant system based on ion channel and application thereof

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Title
ABAD ET AL.: "sequence 6476 from patent US9115368", 《NCBI》 *
BLAKEY ET AL.: "Purification of the Escherichia coli ammonium transporter AmtB reveals a trimeric stoichiometry", 《BIOCHEM. J.》 *
DANIEL SCHWARZ ET AL.: "Cell-free expression profiling of E. coli inner membrane proteins", 《PROTEOMICS》 *
EMMANUELE SEVERI ET AL.: "The conserved carboxy-terminal region of the ammonia channel AmtB plays a critical role in channel function", 《MOLECULAR MEMBRANE BIOLOGY》 *
JEAN-DENIS PEDELACQ ET AL.: "Engineering and characterization of a superfolder green fluorescent protein", 《NATURE BIOTECHNOLOGY》 *
LUCIANO F. HUERGO ET AL.: "Ternary complex formation between AmtB, GlnZ and the nitrogenase regulatory enzyme DraG reveals a novel facet of nitrogen regulation in bacteria", 《MOLECULAR MICROBIOLOGY》 *
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