CN110904138A - Escherichia coli soluble protein expression vector of Msyb fusion tag, construction method and application - Google Patents
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Abstract
The invention belongs to the technical field of protein engineering, and particularly relates to an escherichia coli soluble protein expression vector of an Msyb fusion tag, a construction method and application. The Msy b fusion tag escherichia coli soluble protein expression vector prepared by the construction method of the Msy b fusion tag escherichia coli soluble protein expression vector can well improve the expression quantity of the target protein and the solubility of the target protein, the target protein soluble expression lays a foundation for protein separation and purification and protein activity research, and the Msy fusion tag escherichia coli soluble protein expression vector can be widely applied to the biological fields of genetic engineering, protein engineering and the like.
Description
Technical Field
The invention belongs to the technical field of protein engineering, and particularly relates to an escherichia coli soluble protein expression vector of an Msyb fusion tag, a construction method and application.
Background
Escherichia coli has the characteristics of thorough knowledge of genetic traits, fast growth, economic culture, high expression level, a plurality of plasmids to be selected and the like, becomes a preferred expression system in the technical field of genetic engineering, and is also the expression system which is developed earliest in the gene expression technology and is applied most widely at present. However, for various reasons, the expression system of E.coli has its own limitations, and when the expressed protein is a complex eukaryotic protein, the E.coli often cannot fold the translated polypeptide chain correctly and cannot form a protein with a native conformation, but forms an insoluble non-functional inclusion body. The folding errors of the polypeptide chains in inclusion bodies require complicated denaturation and renaturation processes to form active proteins, but the success rate is low. The exploration of the soluble expression of the recombinant protein in the escherichia coli has higher academic value and wide application prospect.
Some methods for improving the soluble expression of recombinant proteins in escherichia coli have been found at present, such as engineering bacteria modification, induction expression condition optimization (induction temperature, induction time, inducer concentration and the like), molecular chaperone co-expression, fusion tag addition and the like. The more common method is to use a fusion expression strategy, i.e. the fusion tag and the target protein are connected into a fusion protein for expression, and the use of the fusion tag can promote the soluble expression of the target protein connected with the fusion tag and is convenient for the purification of the target protein. There are two categories according to the function of the fusion tag: one is a label that promotes soluble expression of proteins, such as Maltose Binding Protein (MBP), thioredoxin (Trx), small ubiquitin-like modifying protein (SUMO), disulfide-forming protein a (dsba), glutathione transferase (GST), etc.; another class is purification (detection) tags, such as poly-arginine tag (Arg-tag), poly-histidine tag (His-tag), c-myc-tag, S-tag, streptomycin affinity binding tag, FLAG, and the like. However, because of the wide variety of proteins, no universal fusion tag capable of improving the soluble expression of the protein has been found at present.
MseB is an acidic protein in Escherichia coli, and is considered to have the function of inhibiting protein export function deletion mutation in the past, recent research proves that MseB is also a very effective protein soluble tag, the obvious characteristics of the MseB comprise high-efficiency soluble expression of recombinant protein, relatively small molecular weight (124 amino acids), Escherichia coli endogenous protein and the like, and the characteristics open up a very wide prospect for the application of the MseB in the field of biotechnology.
Disclosure of Invention
The first purpose of the present invention is to provide an expression vector for a soluble protein of E.coli having an Msyb fusion tag, which is directed to the disadvantages of the prior art.
For this reason, the above object of the present invention is achieved by the following technical solutions:
an escherichia coli soluble protein expression vector of an Msyb fusion tag, which is named as BL21-pHis-Msyb and is deposited in China general microbiological culture Collection center (Beijing) of China Committee for culture Collection of microorganisms with the deposit number: CGMCC No. 16044.
The second object of the present invention is to provide a method for constructing a soluble protein expression vector of E.coli having an Msyb fusion tag, in view of the disadvantages of the prior art.
For this reason, the above object of the present invention is achieved by the following technical solutions:
a method for constructing an escherichia coli soluble protein expression vector of an Msyb fusion tag comprises the following steps of carrying out PCR amplification by taking an escherichia coli BL2(DE3) genome as a template and SQE ID NO.2 and SQE ID NO.3 as primers to obtain an Msyb gene fragment with a gene sequence of SQE IDNO.1, carrying out double digestion on pET20, connecting the pET20 and the Msyb gene fragment subjected to double digestion through a soft connector to obtain a connection product, carrying out heat shock transformation on the connection product to DH5 α competent cells, screening recombinant clones to obtain the escherichia coli soluble protein expression vector of the Msyb fusion tag;
the gene sequences are respectively as follows:
SQE ID NO.1：
SEQ ID NO.2：
taagaaggagatatacatatgCACCACCACCACCACCACATGACCATGTACGCAACGCTT
SEQ ID NO.3：
acggagctcgaattcggatccACCACCAGAACCACCACGTTCATCCCACTCATCAGCT。
while adopting the above technical scheme, the present invention can also adopt or combine the following further technical schemes:
preferably, the subdued linker is GGSGG, and the subdued linker is small in molecular weight, polar and hydrophilic.
Preferably, the GenBank accession number GI of the genome of escherichia coli BL2(DE3) is 802133627.
Preferably, pET20 is double digested with NdeI and BamHI.
The invention also aims to provide an application of the escherichia coli soluble protein expression vector of the Msyb fusion tag in expression of fusion proteins, aiming at the defects in the prior art.
For this reason, the above object of the present invention is achieved by the following technical solutions:
use of an e.coli soluble protein expression vector according to the aforementioned Msyb fusion tag for the expression of fusion proteins.
While adopting the above technical scheme, the present invention can also adopt or combine the following further technical schemes:
preferably, the target gene is cloned to a multiple cloning site of an escherichia coli soluble protein expression vector of the Msyb fusion tag, and the target protein and the fusion tag are subjected to fusion expression through induction of isopropyl thiogalactoside to form an expression fusion protein.
Preferably, the expression fusion protein is expressed via Ni2+-NTA column affinity purification.
The invention provides an escherichia coli soluble protein expression vector of an Msyb fusion tag, a construction method and application thereof, the escherichia coli soluble protein expression vector of the Msyb fusion tag prepared by the construction method of the escherichia coli soluble protein expression vector of the Msyb fusion tag can well improve the expression quantity of target protein and the solubility of the target protein, the soluble expression of the target protein lays a foundation for protein separation and purification and protein activity research, and the escherichia coli soluble protein expression vector can be widely applied to the biological fields of genetic engineering, protein engineering and the like.
Drawings
FIG. 1 is a schematic diagram of the construction of BL 21-pHis-Mspy fusion tag E.coli isopropyl thiogalactoside (IPTG) induced soluble expression vector;
wherein:
AP is an ampicillin resistance gene;
f1origin is an origin of replication;
MCS multiple cloning sites include NdeI, BamHI, EcoRI, SacI, SalI, HindIII, NotI, XhoI restriction sites.
FIG. 2 shows the result of double restriction enzyme identification of the constructed vector BL 21-pHis-Msyb;
wherein: m: DNA marker DL 2000; 1: BL 21-pHis-Mspy map of two-enzyme digestion.
FIG. 3 is a schematic diagram of BL 21-pHis-Mscyb-cTnI expression vector construction;
wherein: cTnI is the human troponin I gene.
FIG. 4 shows the double restriction enzyme identification result of the constructed vector BL 21-pHis-Msyb-cTnI;
wherein: m: DNA marker DL 2000; 1: pHis-Msyb-cTnI double-enzyme cutting electrophoresis picture.
FIG. 5 shows the difference between the expression results of BL 21-pHis-Mscyb expression vector and pET20 expression vector for human cTnI protein;
wherein: m: protein molecular weight standards (molecular weights of 97.2kD, 66.4kD, 44.3kD, 29.0kD and 20.1kD, respectively); 1: pET20-cTnI post-induction supernatant; 2: precipitation after pET20-cTnI induction; 3: pHis-Msyb-cTnI post-induction supernatant; 4: precipitation after induction of pHis-Msyb-cTnI.
FIG. 6 is an electrophoretogram of BL 21-pHis-Mscyb vector expressed human cTnI after affinity chromatography purification;
wherein: m: protein molecular weight standards (molecular weights of 97.2kD, 66.4kD, 44.3kD, 29.0kD and 20.1kD, respectively); 1: purified human cTnI.
Detailed Description
The invention is described in further detail with reference to the figures and specific embodiments.
The specific experimental conditions not specified in the examples are conventional experimental conditions, such as those described in the handbook of molecular cloning experiments, or experimental methods recommended by the manufacturer of the reference kits.
Before the examples are described, a brief description will be given of some of the raw biological reagents used in the examples:
pET20 plasmid, purchased from Novagen;
coli DH5 α, gene cloning host bacteria, Shanghai Biotechnology Co., Ltd;
coli BL21(DE3), protein expression host bacteria, product of shanghai bio-technology limited;
in the examples, sequencing of related genes and synthesis of primers were performed by Nanjing Kinshire Biotech;
NdeI (FD0583) BamHI (FD0054) XhoI (FD 0694), Thermo Fisher Scientific product;
PCR amplification enzyme (2 × ES Taq MasterMix, Huntington Nongyang Biotechnology Co., CW0690A/5 × 1ml),
plasmid DNA small extraction kit, DNA recovery kit, DNA glue recovery kit, Baosheng bioengineering (Dalian) Co., Ltd product;
clonexpress II One Step Cloning Kit (C112-01/02), a product of Biotechnology Inc. of Nanjing Novowed;
ampicillin (Amp), a product of shanghai bio-technology limited;
DNA molecular quality standard, protein molecular quality standard, and Baosheng bioengineering (Dalian) product.
Example 1: construction of fusion tag escherichia coli isopropyl thiogalactoside (IPTG) induced soluble expression vector
Coli BL21(DE3) was inoculated in LB liquid medium at 1 ‰ inoculum size, cultured overnight at 37 ℃ at 200rpm, and cultured overnight with Baosheng biological plasmid DNA miniprep extraction kit.Mseb primers SEQ ID NO.2 and SEQ ID NO.3 were designed with reference to GenBank accession number GI: 802133627 of E.coli genome sequence, PCR amplification was performed with E.coli BL21(DE3) genome as template and SEQ ID NO.2 and SEQ ID NO.3 as primers, the PCR amplification product was purified and recovered to obtain Mseb gene fragment SEQ ID NO. 1. plasmid T20 was double digested with NdeI and BamHI, 1% gel electrophoresis was performed, the digestion product was recovered with DNA gel recovery Kit, the digestion products of SEQ ID NO.1 and pET20 were ligated with Clonexpress II One StepConing Kit, the ligation product was transformed into DH5 α competent cells, which were named as 100ug/ml antibiotic-containing LB agar medium, recombinant plasmid was screened, and verified by PCR for correct cloning and PCR sequencing and PCR-78-36yb plasmid.
SQE ID NO.1：
(1) The primer sequences are as follows:
SEQ ID NO.2：
taagaaggagatatacatatgCACCACCACCACCACCACATGACCATGTACGCAACGCTT
SEQ ID NO.3：
acggagctcgaattcggatccACCACCAGAACCACCACGTTCATCCCACTCATCAGCT
(2) PCR product amplification and recovery of DNA fragment of interest
The PCR amplification system was performed according to the instructions.
PCR amplification procedure: denaturation at 95 ℃ for 5min, cycling conditions: 30s at 95 ℃, 30s at 54 ℃ and 25s at 72 ℃ for 30 cycles, and finally extension at 72 ℃ for 10 min. The gene amplification instrument is a Hangzhou Bori Life ECO gene amplification instrument TC-96/G/H (b).
And (3) recovering PCR amplification products: the DNA recovery kit of Boehringer Bioengineering (Dalian) Co., Ltd. was used according to the instructions.
(3) pET20 double enzyme digestion and enzyme digestion product recovery
50ul of digestion system, NdeI 4ul, BamHI 4ul, pET20 plasmid 4ug, digestion buffer 5ul, using ddH2Adding O to 50ul, cutting at 37 deg.C for 3h, performing 1% agarose gel electrophoresis, and separatingAnd (3) recovering the DNA glue recovery reagent kit of the Bosheng bioengineering (Dalian) company Limited.
(4) Join, convert, and screen
Msyb gene fragment and pET20 enzyme digestion product concentration are measured by an ultramicro spectrophotometer NanoDrop 2000, the two are connected by a Cloneexpress II One Step Cloning Kit according to a molar ratio of 3:1, the connection condition is 37 ℃ for 30min, the product is connected with an ice bath for 5min, 100ul DH5 α competent cells are added, the ice bath is 30min, the mixture is immediately subjected to water bath hot shock at 42 ℃ for 90s and is subjected to ice bath for 2min, the mixture is coated on an LB agar culture medium containing 100ug/ml ampicillin, the mixture is subjected to inversion culture overnight at 37 ℃ for recombinant clone screening, a positive clone is selected and inoculated into an LB liquid culture medium containing aminobenzyl antibiotic, the mixture is cultured at 37 ℃ and 250rpm for 5h, a Booshenbio plasmid DNA small-extraction Kit is used for extracting plasmids, PCR amplification is carried out by taking SEQ ID NO.2 and SEQ ID NO.3 as primers, the amplification condition is 95 ℃ denaturation 5min, the cycle condition is named as 95 ℃ for 30s at 95 ℃, the cycle is named as pH30 s at 54 ℃ and 72 ℃, the cycle is preserved in 72 ℃, the China society for preservation, the China, the extension study of the Beijing university institute of Escherichia coli DNA, the Beijing university institute, the Escherichia coli DNA, the PCR is carried out the restriction enzyme digestion system, the restriction enzyme digestion of the China-10, the CGMCC No. 7 is carried out, the CGMCC No. 7, the restriction enzyme digestion system, the CGMCC 3, the restriction enzyme digestion system is carried out, the.
Referring to FIG. 1, FIG. 1 is a schematic diagram of the construction of BL 21-pHis-Mspb fusion tag Escherichia coli isopropyl thiogalactoside (IPTG) induced soluble expression vector;
wherein:
AP is an ampicillin resistance gene;
f1origin of replication;
MCS multiple cloning sites include NdeI, BamHI, EcoRI, SacI, SalI, HindIII, NotI, XhoI restriction sites.
Referring to FIG. 2, FIG. 2 shows the result of double-restriction enzyme identification of the constructed vector BL 21-pHis-Mseb;
wherein: m: DNA marker DL 2000; 1: BL21-pHis-Msyb double-enzyme cutting electrophoresis picture, the strip of about 3700bp is pET20 carrier frame, the strip of about 450bp is Mysb gene.
Example 2: fusion expression of human cTnI gene and Msyb
2.1 construction of human cTnI-Mscyb expression vector
Human cTnI gene is used as a template (synthesized by Nanjing Kinsley biotechnology company, shown as SEQ ID NO.4), and SEQ ID NO.5 and SEQ ID NO.6 are used as primers for PCR amplification, wherein the amplification conditions are as follows: denaturation at 95 ℃ for 5min, cycling conditions: 30s at 95 ℃, 30s at 55 ℃ and 25s at 72 ℃ for 28 cycles, finally extending for 10min at 72 ℃, carrying out 1% agarose gel electrophoresis, and purifying the PCR product by using a DNA gel recovery kit of Boehringer bioengineering (Dalian) Co.
SEQ ID NO.4：
SEQ ID NO.5：
ggtggttctggtggtggatccATGGCGGATGGGAGCAGC
SEQ ID NO.6：
gtggtggtggtggtgctcgagTCAGCTCTCAAACTTTTTCTTGCG
Taking BL 21-pHis-Mspb 4ug, 50ul enzyme digestion system, BamH 4ul, XhoI 4ul, enzyme digestion buffer 5ul, using ddH2Supplementing O to 50ul, carrying out enzyme digestion at 37 ℃ for 3h, carrying out 1% agarose gel electrophoresis, and purifying the enzyme digestion vector by using a DNA gel recovery kit of Boehringer bioengineering (Dalian) Co.
The cTnI gene and BL21-pHis-Msyb enzyme digestion vector are connected by Clon express II One StepCringing Kit in a molar ratio of 3:1, the connection condition is 30min at 37 ℃, the connection product is subjected to ice bath for 5min, 100ul DH5 α competent cells are added, ice bath is carried out for 30min, 42 ℃ water bath hot shock is immediately carried out for 90s, ice bath is carried out for 2min, the mixture is coated on LB agar culture medium containing 100ug/ml ampicillin, inverted culture is carried out overnight at 37 ℃, positive clones are selected and inoculated into LB liquid culture medium containing aminobenzyl antibiotic, the plasmid is extracted after culture at 37 ℃ and 250rpm for 5h, PCR amplification is carried out by taking SEQ ID NO.5 and SEQ ID NO.6 as primers, the amplification condition is 5min at 95 ℃, the cycle condition is 30s at 95 ℃, 25s at 55 ℃, 25s at 72 ℃ for 28 cycles, finally, extension is carried out for 10min at 72 ℃, the amplification is named as BamHI and XhoI double enzyme digestion, meanwhile, southern King enzyme digestion, 25s sequencing, PCR and Tnyb gene amplification are carried out, and TnI-21-TnI complete PCR verification.
Referring to FIG. 3, FIG. 3 is a schematic diagram of BL 21-pHis-Mscyb-cTnI expression vector construction;
wherein: cTnI is the human troponin I gene.
Referring to FIG. 4, FIG. 4 shows the double restriction enzyme identification result of the constructed vector BL 21-pHis-Mscyb-cTnI;
wherein: m: DNA marker DL 2000; 1: pHis-Mscyb-cTnI double-enzyme cutting electrophoresis picture, about 4200bp strip is BL 21-pHis-Mscyb vector framework, and about 630bp strip is cTnI gene.
2.2 human cTnI-Mscyb protein expression and purification
BL21-pHis-Msyb-cTnI expression vector is transformed into E.coli BL21(DE3) competent cells by a heat shock method (ice bath for 30min, water bath heat shock at 42 ℃ immediately for 90s, ice bath for 2min), the competent cells are spread on LB agar medium containing 100ug/ml ampicillin, inverted and cultured at 37 ℃ overnight, a single colony is selected and inoculated to LB liquid medium containing ampicillin, and the strain is preserved after culture at 37 ℃ and 250rpm for 7 h.
Recovering strain BL21-pHis-Msyb-cTnI, inoculating the strain preserved in refrigerator at-80 deg.C to 50ml LB (ampicillin) culture medium at 1 ‰, shaking, and culturing at 37 deg.C 200rpm overnight.
1% of the inoculum was inoculated into 1L LB (ampicillin) medium in shake flasks, incubated at 37 ℃ at 250rpm for about 3h to an OD600 of 0.5-0.6, and Isopropylthiogalactoside (IPTG) was added to a final concentration of 1mM and induced at 37 ℃ at 250rpm for 4 h.
Centrifuging to collect thalli and break escherichia coli cells, subpackaging the thalli in a centrifuge cup, centrifuging at 8000rpm for 10min, discarding supernatant, resuspending the thalli with 50mM Tris and 0.3NaCL buffer solution with pH8.0, adding 1mM PMSF with final concentration to carry out ice-bath ultrasonic breaking, wherein the ultrasonic breaking conditions are as follows: sonication for 4s, 6s intervals, total sonication time 30min, sonication power 150W, disruption solution 12000rpm, centrifugation at 4 ℃ for 30min, SDS-PAGE showing soluble cTnI-Msyb supernatant, see lane 3 in FIG. 5.
Referring to FIG. 5, FIG. 5 shows the difference between the expression results of BL 21-pHis-Mscyb expression vector and pET20 expression vector for human cTnI protein;
wherein: m: protein molecular weight standards (molecular weights of 97.2kD, 66.4kD, 44.3kD, 29.0kD and 20.1kD, respectively); 1: pET20-cTnI post-induction supernatant; 2: precipitation after pET20-cTnI induction; 3: pHis-Msyb-cTnI post-induction supernatant; 4: precipitation after induction of pHis-Msyb-cTnI. It is known that pET20 expresses human cTnI protein in the form of inclusion body, and BL 21-pHis-Mscyb expresses human cTnI protein in the form of soluble.
And (3) purification: taking the supernatant and passing the supernatant through Ni2+-NTA column purification of target protein, loading buffer 50mM Tris, 0.3M NaCl pH8.0, equilibration buffer 50mM Tris, 0.3M NaCl, 50mM imidazole pH8.0, elution buffer 50mM Tris, 0.3M NaCl, 500mM imidazole pH8.0, dialyzing the eluted target protein in dialysis buffer for 24h, changing solution once for 12h, dialyzing buffer 50mM Tris, 0.2M NaCl, 2mM EDTA pH8.0, SDS-PAGE after dialysis showing that cTnI-Msyb protein purification is more than 90%, see lane No.1 in figure 6.
Referring to FIG. 6, FIG. 6 is an electrophoretogram of BL 21-pHis-Mscyb vector expressed human cTnI after affinity chromatography purification;
wherein: m: protein molecular weight standards (molecular weights of 97.2kD, 66.4kD, 44.3kD, 29.0kD and 20.1kD, respectively); 1: purified human cTnI.
2.3 detection of human cTnI Activity
And (3) measuring the protein concentration of the purified cTnI-Msyb by a Coomassie brilliant blue method, diluting by 50000 times, and then carrying out activity detection by a Beckmann ACCESS2 chemiluminescence immunoassay analyzer, wherein the detection concentration is 10.21ng/ml, and the protein activity rate is 68.07% (original 0.7 mg/ml).
The experiments show that the soluble expression of the target protein can be realized by using the BL 21-pHis-Mseb vector to express the recombinant protein, and the method can be widely applied to the research of the soluble expression of the recombinant protein.
The above detailed description is to be construed as illustrative of the present invention, and is intended to be exemplary, rather than limiting, of the invention, and it is intended to cover all modifications, equivalents, and improvements made by the present invention within the spirit and scope of the appended claims.
Claims (8)
1. An escherichia coli soluble protein expression vector of an Msyb fusion tag, which is named as BL21-pHis-Msyb and is deposited in China general microbiological culture Collection center (Beijing) of China Committee for culture Collection of microorganisms with the deposit number: CGMCC No. 16044.
2. A construction method of an escherichia coli soluble protein expression vector of an Msyb fusion tag is characterized by comprising the following steps of carrying out PCR amplification by taking an escherichia coli BL2(DE3) genome as a template and SQE ID NO.2 and SQE ID NO.3 as primers to obtain an Msyb gene fragment with a gene sequence of SQE ID NO.1, carrying out double enzyme digestion on pET20, connecting pET20 and the Msyb gene fragment subjected to double enzyme digestion through a soft connector to obtain a connection product, carrying out heat shock transformation on the connection product to DH5 α competent cells and screening recombinant clones to obtain the escherichia coli soluble protein expression vector of the Msyb fusion tag;
the gene sequences are respectively as follows:
SQE ID NO.1：
SEQ ID NO.2：
taagaaggagatatacatatgCACCACCACCACCACCACATGACCATGTACGCAACGCTTSEQ ID NO.3：
acggagctcgaattcggatccACCACCAGAACCACCACGTTCATCCCACTCATCAGCT。
3. the method for constructing an escherichia coli soluble protein expression vector of an Msyb fusion tag according to claim 2, wherein the gentle linker is GGSGG.
4. The method for constructing an escherichia coli soluble protein expression vector of an Msyb fusion tag according to claim 2, wherein GenBank accession number GI of the genome of escherichia coli BL2(DE3) is 802133627.
5. The method for constructing an Msyb fusion tagged E.coli soluble protein expression vector according to claim 2, wherein pET20 is digested with NdeI and BamHI in two steps.
6. Use of an escherichia coli soluble protein expression vector of the Msyb fusion tag of claim 1 for expressing a fusion protein.
7. The Mryb fusion tag E.coli soluble protein expression vector of claim 6, wherein the Mryb fusion tag E.coli soluble protein expression vector is used for expressing fusion protein, and the Mryb fusion tag E.coli soluble protein expression vector is used for expressing fusion protein by cloning a target gene to a multiple cloning site of the Mryb fusion tag E.coli soluble protein expression vector and inducing with isopropylthiogalactoside.
8. Use of the Msyb fusion tag E.coli soluble protein expression vector of claim 7 for expression of fusion proteins via Ni2+-NTA column affinity purification.
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