CN117384930A - Construction method of recombinant escherichia coli engineering strain for expressing cat granulocyte colony stimulating factor - Google Patents
Construction method of recombinant escherichia coli engineering strain for expressing cat granulocyte colony stimulating factor Download PDFInfo
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Abstract
A construction method of recombinant escherichia coli engineering strain expressing cat granulocyte colony stimulating factor belongs to the field of recombinant engineering strain, and comprises the following steps: synthesizing rfG-CSF optimized gene; double-enzyme digestion is carried out on the prepared pUC57-rfG-CSF plasmid and pCold I plasmid, and the recombinant prokaryotic expression plasmid is constructed after the connection of the recovered and purified target fragment and the competent cell of E.coli Rosetta (DE 3) expression is transformed; after the recombinant escherichia coli engineering strain identified correctly is induced to be expressed, the bacterial liquid is crushed by ultrasound, and the supernatant and the sediment are collected by centrifugation; washing, dissolving and purifying the obtained rfG-CSF inclusion body to obtain rfG-CSF stock solution; the purified rfG-CSF stock was subjected to gradient dialysis renaturation. The recombinant cat granulocyte colony stimulating factor expressed by the invention has good biological activity, large expression quantity, short fermentation culture period of recombinant strain and low cost.
Description
Technical Field
The invention belongs to the technical field of recombinant engineering strains, and particularly relates to a construction method of a recombinant escherichia coli engineering strain for expressing a cat granulocyte colony stimulating factor.
Background
Granulocyte colony-stimulating factor (G-CSF) is a cytokine that promotes neutrophil growth. Granulocyte colony-stimulating factor (G-CSF) has 5 cysteine residues, of which 4 form two pairs of disulfide bonds between Cys36 and Cys42, cys74 and Cys64, cys17 being unpaired cysteines, a factor necessary for maintaining the biological function of G-CSF (C P Hill, T D Osslund, deisenberg.the structure ofgranulocyte-color-stimulating factor and its relationship to other growth factors.proc natalacad Sci U s.1993, 90 (11): 5167-71.). The recombinant rhG-CSF expression level, biological activity and stability are further improved by the researcher to optimize the human G-CSF sequence by escherichia coli preference codons.
The feline granulocyte colony-stimulating factor (fG-CSF) is a glycoprotein molecule consisting of 174 amino acid residues produced by monocytes, fibroblasts and endothelial cells, and has high amino acid sequence homology with other species, canine (90.8%) and human (87.4%). Recombinant feline granulocyte colony-stimulating factor (rfG-CSF) has multiple drug functions including: treating leucopenia caused by infection of multiple pathogens such as cat virus (cat plague, cat leukemia, cat hepatitis, etc.), bacteria, fungi, parasites, etc.; preventing and treating leukopenia caused by various medicines; can be used for treating leucopenia caused by hematopoietic dysfunction and blood loss of bone marrow, and has wide application prospect.
Foreign recombinant human granulocyte colony-stimulating factor (rhG-CSF) is approved for clinical treatment of neutropenia in companion dogs and cats for a variety of reasons. However, several studies have demonstrated that treatment of dogs and cats with human-derived G-CSF has a serious problem in that specific neutralizing antibodies to rhG-CSF are induced after 3 weeks, neutralizing exogenous and endogenous G-CSF, resulting in neutropenia (Hammond, w.p., csiba, e., canin, a., hockman, h., souza, l.m., layton, j.e., dale, d.c.,1991.Chronic neutropenia:a new canine model induced by human granulocyte colony-stimulating factor.j.clin.invest.87, 704-710.) (Lothrop, c.d., warren, d.j., souza, l.m., et al.1988.correct of canine cyclic hematopoiesis with recombinant human granulocyte colony-stimulating factor 72,1324-1328. Therefore, it is necessary to treat dogs and cats with a recombinant feline granulocyte colony-stimulating factor (rfG-CSF) with species-specific properties to achieve a good effect.
At present, the main technical problems of using escherichia coli as a recombinant protein expression system to express recombinant feline granulocyte colony-stimulating factor (rfG-CSF) are that: the expressed recombinant cat granulocyte colony-stimulating factor (rfG-CSF) has poor biological activity, small expression quantity and low specific activity.
Disclosure of Invention
The invention aims to provide a construction method of recombinant escherichia coli engineering strain for expressing the feline granulocyte colony-stimulating factor, and the recombinant feline granulocyte colony-stimulating factor (rfG-CSF) expressed by the invention has good biological activity, large expression quantity and specific activity of more than 1 multiplied by 10 7 U/mg。
The technical scheme adopted by the invention for solving the technical problems is as follows:
the construction method of the recombinant escherichia coli engineering strain expressing the feline granulocyte colony stimulating factor comprises the following steps:
step one, according to a known cat granulocyte colony stimulating factor gene sequence, performing escherichia coli preference codon optimization by using an optimumGene codon optimization technology, and synthesizing a rfG-CSF optimized gene;
step two, preparing pUC57-rfG-CSF plasmid;
step three, carrying out BamHI and EcoRI double digestion on pUC57-rfG-CSF plasmid and pCold I prokaryotic expression plasmid respectively, recovering and purifying rfG-CSF gene fragment and pCold I vector fragment, and connecting by using T4DNA to construct recombinant prokaryotic expression plasmid pCold I-rfG-CSF, and transforming Escherichia coli (Escherichia coli) Rosetta (DE 3) expression competent cells;
step four, performing induced expression on the identified correct recombinant escherichia coli engineering strain Rosetta (DE 3) (pCold I-rfG-CSF), performing ultrasonic bacterial liquid breaking, centrifuging, collecting supernatant and precipitate, and performing protein expression analysis and identification to obtain rfG-CSF inclusion body;
step five, washing, dissolving and purifying the rfG-CSF inclusion body to obtain rfG-CSF stock solution;
and step six, performing gradient dialysis renaturation on the purified rfG-CSF stock solution.
As a preferred embodiment, the sequence of the rfG-CSF optimized gene is shown in SEQ ID NO.1.
In a preferred embodiment, the specific operation procedure of the second step is as follows: the synthesized rfG-CSF optimized gene is cloned to pUC57 plasmid, and the pUC57-rfG-CSF plasmid is prepared and obtained through enzyme digestion and sequencing identification.
As a preferred embodiment, the specific operation flow of the fourth step is as follows:
inoculating recombinant E.coli engineering strain Rosetta (DE 3) (pCold I-rfG-CSF) with 0.1% inoculum size into LB culture solution, shake culturing at 37deg.C at 200rpm/min overnight, collecting overnight culture, inoculating with 1% inoculum size into LB culture solution, shake culturing at 37deg.C at 200rpm/min until A 600 When the temperature reaches 0.5, cooling the culture solution to 15 ℃, standing for at least 30min, adding IPTG to a final concentration of 0.5mM at 25 DEG CInducing expression at 170rpm/min for 16h, centrifuging at 5000rpm/min for 5min to collect bacterial liquid, re-suspending with PBS, ultrasonically crushing bacterial liquid in ice bath, centrifuging at 12000rpm/min for 10min to collect supernatant and precipitate.
As a preferred embodiment, the LB medium contains 100. Mu.g/ml of Amp.
In a preferred embodiment, the specific operation procedure of the fifth step is as follows:
rfG-CSF inclusion bodies were washed several times with inclusion body washing solution, and then rfG-CSF inclusion bodies were dissolved overnight at 4℃with inclusion body dissolution solution, centrifuged at 15000rpm/min for 15min to obtain supernatants, and purified using Ni Focus 6FF (IMAC) affinity chromatography column.
As a preferred embodiment, the inclusion body wash solution contains 0.02M PB,0.5M NaCl,2MUrea,1%Triton X-100, pH7.4.
In a preferred embodiment, the inclusion body lysate contains 0.02M PB,0.5M NaCl,8M urea.
In a preferred embodiment, the specific operation procedure in the sixth step is as follows:
diluting the purified rfG-CSF stock solution until the protein concentration is lower than 1mg/ml, filling the diluted stock solution into a dialysis bag, and carrying out gradient dialysis renaturation in renaturation buffer solution.
In a preferred embodiment, the renaturation buffer comprises 0.02M PB,0.5MNaCl,0-6MUREa,5% glycerol, 1% glycine, 0.2%PEG6000,1mM GSH,0.1mM GSSG.
The beneficial effects of the invention are as follows:
in order to construct a recombinant Escherichia coli engineering strain for expressing the feline granulocyte colony-stimulating factor, the invention carries out codon optimization design synthesis on the feline granulocyte colony-stimulating factor gene, clones the recombinant Escherichia coli colony-stimulating factor gene into a prokaryotic expression vector pCold I, constructs a recombinant prokaryotic expression plasmid pCold I-rfG-CSF and converts Escherichia coli (Escherichia coli) Rosetta (DE 3) expression competent cells to carry out induction expression. SDS-PAGE and Westernblotting analysis result shows that the recombinant colibacillus engineering strain constructed by the invention can effectively express recombinant cat granulocyte colony-stimulating factor (rfG-CSF), and its molecular weight is identical to the expected size, about 22.7kDa, mainly usesInclusion body forms exist. Meanwhile, the Ni Focus 6FF (IMAC) affinity chromatographic column is adopted to purify rfG-CSF, after renaturation by a gradient dialysis method, the CCK8/NFS-60 cell proliferation method is used for detecting and analyzing rfG-CSF biological activity, and the result shows that rfG-CSF has good biological activity and ED 50 A value of about 100pg/ml and a specific activity of greater than 1X 10 7 U/mg。
In addition, the recombinant escherichia coli engineering strain has the advantages of short fermentation culture period, relatively low cost and simple and mature process, and is suitable for industrial scale-up production.
Drawings
FIG. 1 shows the results of the expression identification of recombinant feline granulocyte colony-stimulating factor (rfG-CSF). In the figure, a is SDS-PAGE result of a recombinant escherichia coli engineering strain expressed rfG-CSF, wherein M is a protein Marker;1 is non-induced whole bacteria; 2 is the whole bacteria after induction; 3, ultrasonic crushing and precipitation after induction; 4 is the ultrasonic disruption supernatant after induction. b is a Westernblotting result of expressing rfG-CSF for recombinant E.coli engineering strains, wherein M is a protein Marker;1 is non-induced whole bacteria; 2 is the whole bacteria after induction.
FIG. 2 shows the result of SDS-PAGE analysis after purification and renaturation of rfG-CSF. In the figure, M is a protein Marker; 1. 2 are rfG-CSF after renaturation; 3. 4 are purified rfG-CSF.
FIG. 3 is a graph of rfG-CSF stimulated NSF-60 cell proliferation.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Experimental materials
The pCold I prokaryotic expression plasmid was given away by Zhang Xuehan researchers (academy of sciences of agriculture, jiangsu province, veterinary institute); coli (Escherichia coli) Rosetta (DE 3) expressing competent cells were purchased from shanghai virtuous biotechnology limited; ni Focus 6FF (IMAC) affinity chromatography column was purchased from Wuhan Hui Biolabs; recombinant human granulocyte colony-stimulating factor (rhG-CSF) was purchased from Dalian Mey Biotechnology Co., ltd; cell Counting Kit-8 (CCK-8) are available from Biyun Tian Biotechnology Co., ltd; mouse leukemia G-CSF dependent cells (NFS-60 cells) were purchased from Wohsaiplon life technologies Co., ltd; recombinant human granulocyte colony-stimulating factor (G-CSF) was purchased from Meiluno (Mei Lun); protein quantification kit Detergent Compatible Bradford Protein Quantification Kit was purchased from nupran biosystems; the endonucleases BamHI and EcoRI were purchased from TaKaRa, and the other reagents were of imported or domestic analytical purity.
EXAMPLE 1 recombinant feline granulocyte colony-stimulating factor (rfG-CSF) Gene design
According to the published gene sequence of the feline granulocyte colony-stimulating factor (ACCESSION: AB 042552) (AYamamoto, AIwata, KTuchiya, AKatsumata, K Oishi, T Saito, H Tsujimoto, AHasegawa, S Ueda. Molecular cloning and expression ofthe cDNA encoding feline granulocyte colony-stimulating factor. Gene.2001,274: 263-269.) the recombinant feline granulocyte colony-stimulating factor (fG-CSF) optimizing gene (SEQ ID NO. 1) was synthesized by Nanjing gold srey biosystems using the optimumGene codon optimization technique. The synthesized rfG-CSF optimized gene is cloned to pUC57 plasmid, and finally pUC57-rfG-CSF plasmid is obtained through enzyme digestion and sequencing identification.
According to the invention, the optimal gene codon optimization technology is used for optimizing the escherichia coli preference codon of the feline granulocyte colony stimulating factor (fG-CSF), and the algorithm fully considers various complex factors possibly encountered in different stages of protein expression, so that the expression efficiency of recombinant proteins is obviously improved. In addition, 5 codons are subjected to sense mutation, including mutation of 17-bit Cys into Ser, so that the probability of mismatch of intramolecular disulfide bonds and intermolecular disulfide bonds is reduced, the renaturation correct folding rate is improved, and the biological activity and stability of rfG-CSF are enhanced.
EXAMPLE 2 construction of recombinant E.coli engineering Strain and expression of recombinant feline granulocyte colony-stimulating factor (rfG-CSF)
1. The pUC57-rfG-CSF plasmid and pCold I prokaryotic expression plasmid obtained in example 1 were digested with BamHI and EcoRI, respectively, the rfG-CSF gene fragment and pCold I vector fragment were recovered and purified, and then ligated with T4DNA to construct recombinant prokaryotic expression plasmid pCold I-rfG-CSF, which was transformed into E.coli (Escherichia coli) Rosetta (DE 3) expression competent cells.
2. The recombinant E.coli engineering strain Rosetta (DE 3) (pCold I-rfG-CSF) which is identified correctly is inoculated into LB culture solution (containing 100. Mu.g/ml of Amp) according to an inoculum size of 0.1%, and after shaking culture at 37℃and 200rpm/min overnight, the overnight culture is taken and inoculated into LB culture solution (containing 100. Mu.g/ml of Amp) according to an inoculum size of 1%, and shaking culture is carried out at 200rpm/min until A 600 When the temperature reaches 0.5, cooling the culture solution to 15 ℃, standing for more than 30min, adding IPTG to a final concentration of 0.5mM, inducing expression at 170rpm/min for 16h, collecting bacterial liquid by centrifugation at 5000rpm/min for 5min, re-suspending by PBS, ultrasonically crushing the bacterial liquid in an ice bath, centrifugally collecting supernatant and precipitate at 12000rpm/min for 10min, and detecting the expression condition of recombinant cat granulocyte colony-stimulating factor (rfG-CSF) by SDS-PAGE and Westernblotting methods.
The recombinant prokaryotic expression plasmid pCold I-rfG-CSF is transformed into Escherichia coli (Escherichia coli) Rosetta (DE 3) expression competent cells, the correct recombinant Escherichia coli engineering strain Rosetta (DE 3) (pCold I-rfG-CSF) is identified for induced expression, bacterial liquid is crushed by ultrasound, supernatant and sediment are collected by centrifugation, and protein expression analysis and identification are carried out. SDS-PAGE results (FIG. 1 a) show that recombinant E.coli engineering strain Rosetta (DE 3) (pCold I-rfG-CSF) after induction expressed a specific protein band of about 22.7kDa (SEQ ID NO. 2) consistent with the expected size, mainly in inclusion form, and that the uninduced strain was not expressed. Westernblotting analysis (FIG. 1 b) shows that recombinant E.coli engineering strain Rosetta (DE 3) (pCold I-rfG-CSF) can induce expression of recombinant feline granulocyte colony-stimulating factor (rfG-CSF).
EXAMPLE 3 purification and renaturation of recombinant feline granulocyte colony-stimulating factor (rfG-CSF)
1. rfG-CSF inclusion bodies expressed by recombinant E.coli engineering strain Rosetta (DE 3) (pCold I-rfG-CSF) were washed 3 times with inclusion body washing solution (0.02M PB,0.5MNaCl,2M Urea,1%Triton X-100pH7.4), followed by overnight dissolution of rfG-CSF inclusion bodies with inclusion body dissolution solution (0.02M PB,0.5M NaCl,8M urea) at 4 ℃, centrifugation at 15000rpm/min for 15min to collect supernatants, purification using Ni Focus 6FF (IMAC) affinity chromatography column, purification steps were referred to the operating instructions.
2. The purified rfG-CSF stock solution was diluted to a protein concentration of less than 1mg/ml, and then placed in a dialysis bag, and subjected to gradient dialysis renaturation in renaturation buffer (0.02M PB,0.5M NaCl,Urea (6M, 4M, 2M, 0M), 5% glycerol, 1% glycine, 0.2%PEG6000,1mM GSH,0.1mM GSSG). The Bradford method detects rfG-CSF protein concentration and SDS-PAGE detects rfG-CSF purification and renaturation results.
The rfG-CSF inclusion body induced and expressed by the recombinant escherichia coli engineering strain Rosetta (DE 3) (pCold I-rfG-CSF) is subjected to washing, dissolving, purifying and dialyzing renaturation operation to obtain rfG-CSF stock solution. SDS-PAGE detection results (figure 2) show that the expressed rfG-CSF inclusion body has higher purity after being washed by inclusion body washing liquid for 3 times, the purity is further improved after being purified by using an Ni Focus 6FF (IMAC) affinity chromatography column after dissolution and denaturation, after renaturation, the purity is further improved by centrifugation at 12000rpm/min for 10min, and supernatant is collected for SDS-PAGE analysis, so that rfG-CSF stock solution with higher purity is obtained, and the concentration of protein stock solution after detection and renaturation reaches 0.218mg/ml.
EXAMPLE 4 analysis of the biological Activity of recombinant feline granulocyte colony-stimulating factor (rfG-CSF)
rfG-CSF stock, rhG-CSF reference (from Dalian Mei Biotechnology Co., ltd., batch MA 0605), rfG-CSF control (from Beijing Bo Ledeb Biotechnology Co., ltd., batch 20210101) were each serially diluted 10-fold with RPMI1640 basal medium in 96-well plates for a total of 5 dilutions (1 pg/ml,10pg/ml,100pg/ml,1000pg/ml,10000 pg/ml), each dilution being provided with 3 replicate wells, 50. Mu.l/well; regulating NFS-60 cell concentration to 2.0X10 5 Cell suspensions were seeded into the serial diluted 96-well plates, 50 μl/well, 37 ℃,5% co 2 Culturing under the condition for 40-48 h, then 100 μl Kong Lijia μl CCK-8 solution, placing the culture plate in an incubator, and incubating for 1-4 hh, measuring the absorbance at 450nm by using a microplate reader, and drawing a dose response curve.
The CCK8/NFS-60 cell proliferation assay detects rfG-CSF biological activity after purification and renaturation. The response curve of the expressed rfG-CSF stock solution and the rhG-CSF reference and rfG-CSF control to stimulate NFS-60 cell proliferation is shown in FIG. 3, and the proliferation of NFS-60 cells is enhanced with the increase of the dosage of G-CSF, thereby confirming that the expressed rfG-CSF has good biological activity and ED of rfG-CSF 50 A value of about 100pg/ml and a specific activity of greater than 1X 10 7 U/mg。
The recombinant escherichia coli engineering strain Rosetta (DE 3) (pCold I-rfG-CSF) constructed by the cold shock expression vector pCold I can induce the recombinant feline granulocyte colony stimulating factor (rfG-CSF) to be expressed efficiently at normal temperature, and has good biological activity after inclusion body extraction, purification and renaturation.
The invention discloses a recombinant escherichia coli engineering strain for expressing a feline granulocyte colony stimulating factor and a construction method thereof, and the technical parameters can be properly improved by a person skilled in the art by referring to the content of the specification. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the invention has been described with reference to preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the invention described herein without departing from the spirit or scope of the invention.
Claims (10)
1. The construction method of the recombinant escherichia coli engineering strain for expressing the feline granulocyte colony stimulating factor is characterized by comprising the following steps:
step one, according to a known cat granulocyte colony stimulating factor gene sequence, performing escherichia coli preference codon optimization by using an optimumGene codon optimization technology, and synthesizing a rfG-CSF optimized gene;
step two, preparing pUC57-rfG-CSF plasmid;
step three, the pUC57-rfG-CSF plasmid and pCold I prokaryotic expression plasmid are respectively undergone the process of BamHI and EcoRI double digestion, the rfG-CSF gene fragment and pCold I vector fragment are recovered and purified, and connected by using T4DNA so as to construct recombinant prokaryotic expression plasmid pCold I-rfG-CSF, and the recombinant prokaryotic expression plasmid pCold I-rfG-CSF is converted into Escherichia coli (Escherichia coli)
Rosetta (DE 3) expressing competent cells;
step four, performing induced expression on the identified correct recombinant escherichia coli engineering strain Rosetta (DE 3) (pCold I-rfG-CSF), performing ultrasonic bacterial liquid breaking, centrifuging, collecting supernatant and precipitate, and performing protein expression analysis and identification to obtain rfG-CSF inclusion body;
step five, washing, dissolving and purifying the rfG-CSF inclusion body to obtain rfG-CSF stock solution;
and step six, performing gradient dialysis renaturation on the purified rfG-CSF stock solution.
2. The method for constructing recombinant escherichia coli engineering strain expressing the feline granulocyte colony-stimulating factor as set forth in claim 1, wherein the sequence of the rfG-CSF optimization gene is shown in SEQ ID No.1.
3. The method for constructing a recombinant escherichia coli engineering strain expressing a feline granulocyte colony-stimulating factor as set forth in claim 1, wherein the specific operation procedure of the second step is as follows: the synthesized rfG-CSF optimized gene is cloned to pUC57 plasmid, and the pUC57-rfG-CSF plasmid is prepared and obtained through enzyme digestion and sequencing identification.
4. The method for constructing recombinant escherichia coli engineering strains expressing the feline granulocyte colony-stimulating factor according to claim 1, wherein the specific operation procedure of the fourth step is as follows:
inoculating recombinant E.coli engineering strain Rosetta (DE 3) (pCold I-rfG-CSF) with 0.1% inoculum size into LB culture solution, shake culturing at 37deg.C at 200rpm/min overnight, collecting overnight culture, inoculating with 1% inoculum size into LB culture solution, shake culturing at 37deg.C at 200rpm/min until A 600 When the temperature reaches 0.5, cooling the culture solution to 15 ℃, standing for at least 30min, adding IPTG to a final concentration of 0.5mM, inducing expression at 170rpm/min for 16h at 25 ℃, collecting bacterial liquid at 5000rpm/min by centrifugation for 5min, re-suspending by PBS, ultrasonically crushing the bacterial liquid in ice bath, and collecting supernatant and precipitate by centrifugation at 12000rpm/min for 10 min.
5. The method for constructing a recombinant E.coli engineering strain expressing a feline granulocyte colony-stimulating factor as claimed in claim 4, wherein the LB medium contains 100. Mu.g/ml of Amp.
6. The method for constructing a recombinant escherichia coli engineering strain expressing a feline granulocyte colony-stimulating factor as set forth in claim 1, wherein the specific operation procedure of the fifth step is as follows:
rfG-CSF inclusion bodies were washed several times with inclusion body washing solution, and then rfG-CSF inclusion bodies were dissolved overnight at 4℃with inclusion body dissolution solution, centrifuged at 15000rpm/min for 15min to obtain supernatants, and purified using Ni Focus 6FF (IMAC) affinity chromatography column.
7. The method for constructing a recombinant E.coli engineering strain expressing a feline granulocyte colony-stimulating factor as claimed in claim 6, wherein the inclusion body wash contains 0.02M PB,0.5M NaCl,2M Urea,1%Triton X to 100, pH7.4.
8. The method of claim 6, wherein the inclusion body lysate comprises 0.02M PB,0.5M NaCl,8M urea.
9. The method for constructing a recombinant E.coli engineering strain expressing a feline granulocyte colony-stimulating factor as claimed in claim 6, wherein the specific operation procedure in the sixth step is as follows:
diluting the purified rfG-CSF stock solution until the protein concentration is lower than 1mg/ml, filling the diluted stock solution into a dialysis bag, and carrying out gradient dialysis renaturation in renaturation buffer solution.
10. Recombinant escherichia coli worker expressing feline granulocyte colony-stimulating factor as claimed in claim 9
A method for constructing a strain, characterized in that the renaturation buffer contains 0.02M PB,0.5M NaCl,
0-6M Urea,5% glycerol, 1% glycine, 0.2%PEG6000,1mM GSH,0.1mM GSSG.
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