1.Application of a prediction model in inclusion body refolding.
Ting ZHANG ; Ju-Fang WANG ; Yan-Ye FENG ; Zhong YANG ; Li MA ; Xiao-Ning WANG
Journal of Southern Medical University 2009;29(11):2156-2160
OBJECTIVETo establish a prediction method for the refolding of inclusion bodies and classify refolding types of different inclusion bodies directly from their primary structure to improve the efficiency of high throughput refolding process.
METHODSForty-three recombinant proteins performing important biological functions were expressed in E. coli. The probability of forming inclusion bodies of these proteins was predicted using Harrison's two parameter prediction model based on the proteins' amino acid composition. Subsequently, the proteins from the inclusion bodies were refolded using a double denaturation method that involved washing and denaturation in GdnHCl solution followed by denaturation in Urea solution and refolding through dilution.
RESULTSAll the proteins were detected in the form of inclusion bodies using SDS-PAGE method. The proteins were divided into two types according to the results of both solubility prediction and refolding experiments. Fourteen proteins were predicted to have the dependency of soluble expression. The refolding yields of these inclusion bodies were up to 70%. Twenty-nine proteins were predicted to have the high dependency of insoluble expression, and their refolding yields could be higher than 70% and lower than 60%. Comparison of the characteristics between the proteins with high and low refolding yields showed that the theoretical pI was significantly different (P<0.05).
CONCLUSIONSHarrison's two parameter prediction model has the value for potential application in classification of the inclusion bodies and prediction of solubility of proteins refolded from different inclusion bodies. This a novel method enhances the efficiency of high throughput refolding of inclusion bodies, and suggests that the theoretical pI of the proteins is an important parameter in the prediction of refolding yields.
Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; chemistry ; genetics ; Genetic Vectors ; genetics ; Inclusion Bodies ; chemistry ; Models, Biological ; Protein Refolding ; Recombinant Proteins ; biosynthesis ; genetics
2.Chromatography-assisted refolding of a fusion protein containing multiple disulfide bonds.
Weiquan XIE ; Guifeng ZHANG ; Ling GAO ; Yongdong LIU ; Rong YU ; Zhiguo SU
Chinese Journal of Biotechnology 2010;26(8):1157-1164
To establish a refolding process for the protein fused with 12-peptide of hirudin and reteplase (HV12p-rPA), we developed an anion-exchange chromatography assisted method to form correct disulfide bonds. After evaluating various parameters by orthogonal experiments with Q Sepharose XL as refolding medium, we found that urea gradient, sample loading size and L-Arg concentration were three major factors to affect the refolding outcomes, and urea gradient was critical to the recovery yield. Meanwhile, enzymatic activity of the refolded protein was decreased by the increase of sample loading size, and the optimal concentration of L-Arg in the eluting buffer was 1 mol/L. Thus, a dual-gradient of urea and pH on the anion-exchange chromatography resulted in remarkable increase of specific fibrinolytic and anti-coagulative activities of the refolded protein. Compared with the dilution method for refolding HV12p-rPA, the present approach was more effective and advantageous.
Chromatography, Ion Exchange
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methods
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Disulfides
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chemistry
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Fibrinolytic Agents
;
analysis
;
chemistry
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Hirudins
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analysis
;
chemistry
;
Protein Refolding
;
Recombinant Fusion Proteins
;
analysis
;
chemistry
;
Recombinant Proteins
;
analysis
;
chemistry
;
Tissue Plasminogen Activator
;
analysis
;
chemistry
3.Synthesis, refolding and identification of pharmacological activities of neurotoxin JZTX-XI and R3A-JZTX-XI.
Yupeng CHI ; Meichun DENG ; Yuanyuan WU ; Ji LUO ; Minqiang RONG ; Yiya ZHANG ; Dongyi ZHANG ; Xiongzhi ZENG ; Songping LIANG
Chinese Journal of Biotechnology 2011;27(6):900-908
Kv2.1 channel currents in pancreatic beta-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. Because of its central role in this important physiological process, Kv2.1 channel is a promising target for the treatment of type 2 diabetes. Jingzhaotoxin-XI (JZTX-XI) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Two-microelectrode voltage clamp experiments had showed that the toxin inhibited Kv2.1 potassium currents expressed in Xenopus Laevis oocytes. In order to investigate the structure-function relationship of JZTX-XI, the natural toxin and a mutant of JZTX-XI in which Arg3 was replaced by Ala, were synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. Reverse-phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to monitor the oxidative refolding process of synthetic linear peptides to find the optimal renaturation conditions of these toxins. The experiments also proved that the relative molecular masses of refolded peptides were in accordance with their theoretical molecular masses. RP-HPLC chromatogram of co-injected native and refolded JZTX-XI was a single peak. Under the whole-cell patch-clamp mode, JZTX-XI could completely inhibit hKv2.1 and hNav1.5 channels currents expressed in HEK293T cells with IC50 values of 95.8 nmol/L and 437.1 nmol/L respectively. The mutant R3A-JZTX-XI could also inhibit hKv2.1 and hNav1.5 channel currents expressed in HEK293T cells with IC50 values of 1.22 micromol/L and 1.96 micromol/L respectively. However, the prohibitive levels of R3A-JZTX-XI on hKv2.1 and hNav1.5 channels were reduced by about 12.7 times and 4.5 times respectively, indicating that Arg3 was a key amino acid residue relative to the hKv2.1 channel activity of JZTX-XI, but it is also an amino acid residue correlated with the binding activity of JZTX-XI to hNav1.5 channel. Our findings should be helpful to develop JZTX-XI into a molecular probe and drug candidate targeting to Kv2.1 potassium channel in the pancreas.
Animals
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HEK293 Cells
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Humans
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Insulin-Secreting Cells
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metabolism
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Mutant Proteins
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genetics
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pharmacology
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NAV1.5 Voltage-Gated Sodium Channel
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metabolism
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Neurotoxins
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chemical synthesis
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genetics
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pharmacology
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Protein Refolding
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Shab Potassium Channels
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antagonists & inhibitors
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metabolism
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Sodium Channel Blockers
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pharmacology
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Spider Venoms
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genetics
;
pharmacology
;
Transfection
4.Expression, purification and renaturation of proNGF in Escherichia coli.
Hanmin JIANG ; Xinjun CHAI ; Bing HE ; Juan ZHAO ; Xinda YU
Chinese Journal of Biotechnology 2008;24(3):509-514
Nerve growth factor (NGF) promotes neuronal survival and differentiation and stimulates neurite outgrowth. NGF is synthesized as a precursor-proNGF in vivo. In this paper, a pET-proNGF prokaryocyte expression vector was constructed and transformed into E. coli BL21(DE3)pLysS. The proNGF was expressed in the form of non-active aggregated monomer in E. coli after induction with IPTG. SDS-PAGE revealed the proNGF expression product had a Mr.30.2 kD. Western blotting analysis showed that the protein had good antigenicity. Fusion protein was successfully purified by Ni2+-NTA affinity chromatography and cleaved by Enterokinase and 13.1 mg proNGF was obtained from 100 mL cell culture in a typical experiment. The protein was dialyzed in a redox system containing reduced and oxidized glutathione. RP-HPLC was used to analysis the result of the refolding. The refolded proNGF protein can induce neurite outgrowth of PC12 cells, which indicated that pro-form of NGF we obtained had biological activity.
Escherichia coli
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genetics
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metabolism
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Genetic Vectors
;
genetics
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Humans
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Nerve Growth Factor
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biosynthesis
;
genetics
;
Protein Precursors
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biosynthesis
;
genetics
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Protein Renaturation
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Recombinant Fusion Proteins
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biosynthesis
;
genetics
;
isolation & purification
5.The renaturation and purification of RGD-staphylokinase by gel filtration.
Ao CHENG ; Gang SONG ; Hua-Bo SU ; Min YU ; Yu-Yang LI ; Hou-Yan SONG
Chinese Journal of Biotechnology 2002;18(6):693-697
A recombinant RGD-Staphylokinase(RGD-Sak) with thrombolytic and anti-thrombolytic bifunction was expressed in E. coli. The expression product accumulates as inclusion bodies. In order to obtain active molecule, the RGD-Sak in the inclusion body should be denatured and then renatured. The renaturation of RGD-Sak was performed by gel filtration. Comparing with the traditional way of dilution renaturation, gel filtration way is better than the traditional one, since there are some advantages, such as simple processing, high recovery, low cost and higher purity after renaturation, After renaturation, RGD-Sak was purified by Q-Sepharose FF, and the purity was more than 95%. Analysis of CD spectra showed that the final product from the two renaturation ways have similar CD spectra. It was demonstrated that RGD-Sak molecules proceeded correct refolding through gel filtration or dilution renaturation process.
Chromatography, Gel
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Circular Dichroism
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Metalloendopeptidases
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chemistry
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isolation & purification
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Oligopeptides
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isolation & purification
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Protein Folding
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Protein Renaturation
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Recombinant Fusion Proteins
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chemistry
;
isolation & purification
6.Purification and renaturation of recombinant human Cu, Zn-SOD by metal-chelating affinity chromatography.
Jian-Rong LIU ; Jian-Guo LIU ; Xiao-Yu ZHAO ; Ya-Jun GU
Chinese Journal of Biotechnology 2005;21(6):993-997
Overexpression of recombinant Human Cu, Zn-Superoxide Dismutase (rhCu, Zn-SOD) in E. coli results in the form of insoluble inclusion body. Purity of rhSOD inclusion body was over 80% by isolation and purification. After preliminary renaturation by conventional dilution or dialysis, enzyme preparations was respectively purified by using Copper Metals-Chelating Affinity Chromatography (Copper-MCAC). RhSOD specific activity purified by MCAC (from the sample renatured partly by dialysis) was 2.2 times as much as that by dialysis and protein recovery was 64%. RhSOD specific activity purified by MCAC (from the sample renatured partly by dilution) was 5.3 times as much as that by dilution and protein recovery was 25%. The two rhSOD preparations purified by MCAC had specific activities about 5000 u/mg and activity recoveries were all over 130% of the enzyme activities in the samples renatured partly by dilution or dialysis. The above-mentioned results indicated that Copper-MCAC resulted in a purification and further renaturation of target protein. SDS-PAGE showed that the target protein rhSOD (19 kD) was purified homogeneously and NBT activity identification proved that the purified and renatured rhSOD had very strong SOD activity. In conclusion, Copper Metals-Chelaing Affinity Chromatography appears to be a simple, rapid and efficient procedure for purifying and further renaturing rhCu, Zn-SOD by dilution or dialysis. The method provided a new idea for purifying and renaturing recombinant proteins expressed in the form of inclusion body in E. coli.
Chelating Agents
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chemistry
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Chromatography, Affinity
;
methods
;
Escherichia coli
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genetics
;
metabolism
;
Humans
;
Inclusion Bodies
;
genetics
;
Protein Renaturation
;
Recombinant Proteins
;
biosynthesis
;
genetics
;
isolation & purification
;
Superoxide Dismutase
;
biosynthesis
;
genetics
7.Studies on the optimal expression condition, purification and its characterization of ScFv-2F3.
Yuan-Ming LUO ; Ying MU ; Jing-Yan WEI ; Gang-Lin YAN ; Gui-Min LUO
Chinese Journal of Biotechnology 2002;18(1):74-78
The expression vectors of the gene encoding ScFv-2F3 were transformed into E. coli BL21(DE3). Clones of higher expression were first selected, then were grown in the presence of IPTG at 37 degrees C to induce its expression. The culture conditions were carefully optimized. It was found that optimal conditions were as follows: the induction was started as OD590 reached to 1.0-1.8; the concentration of IPTG was 0.3-0.5 mmol/L and induction time is 7 h. The yield of ScFv-2F3 expressed in the selected clones is about 20% of the total proteins. The optimal culture conditions were successfully applied to fermenter of 50 L. The conditions of washing the inclusion bodies were also optimized. A two-step method was used to renature the inclusion body. The expression product of interest and its biological activities were characterized with Western blotting and ELISA. A novel selenium-containing single-chain abzyme with GPX activity was prepared.
Antibodies, Catalytic
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biosynthesis
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chemistry
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genetics
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isolation & purification
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Bioreactors
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microbiology
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Cloning, Molecular
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Escherichia coli
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Gene Expression
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Immunoglobulin Fragments
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biosynthesis
;
chemistry
;
genetics
;
isolation & purification
;
Inclusion Bodies
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metabolism
;
Protein Folding
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Protein Renaturation
;
Recombinant Proteins
;
biosynthesis
;
chemistry
;
genetics
;
isolation & purification
;
Selenium
;
metabolism
8.Expression and renaturation of a novel human single-chain Fv antibody against SARS-CoV.
Jin-Zhu DUAN ; Cai QI ; Wei HAN ; Zhan-Hui WANG ; Gang JIN ; Xi-Yun YAN
Chinese Journal of Biotechnology 2005;21(5):692-697
A novel human ScFv H12 against SARS-CoV has been selected from a SARS immune library. In order to produce a large amount of ScFv H12, pET28a-H12 expression vector was constructed and ScFv H12 was expressed at yield about 30% of total proteins in E. coli . Here two different refolding procedures were used to refold ScFv H12 from inclusion body: gel filtration chromatography and dilution. The results showed that ScFv H12 could be efficiently refolded by both procedures. However, the refolding via gel filtration was 1.5 time more effective than that of dilution. The affinity of ScFv H12 to SARS-CoV virion was detected as Kd = 73.5 nmol/mL.
Antibodies, Monoclonal
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biosynthesis
;
genetics
;
Antibodies, Viral
;
immunology
;
Escherichia coli
;
genetics
;
metabolism
;
Humans
;
Immunoglobulin Fragments
;
biosynthesis
;
genetics
;
immunology
;
Immunoglobulin Variable Region
;
biosynthesis
;
genetics
;
immunology
;
Inclusion Bodies
;
genetics
;
immunology
;
Protein Renaturation
;
Recombinant Proteins
;
biosynthesis
;
chemistry
;
immunology
;
SARS Virus
;
immunology
9.Renaturation, purification and antigenicity identification of recombinant protein of Cysticercus cellulosae expressed in Escherichia coli.
Shaohua ZHANG ; Wanzhong JIA ; Xuenong LUO ; Zhizhong JING ; Guohua WU ; Yadong ZHENG ; Aijiang GUO ; Xuepeng CAI
Chinese Journal of Biotechnology 2008;24(8):1490-1495
To obtain the recombinant 18 kD protein with high purity and normal bioactivity of Cysticercus cellulosae (rCE18), E. coli cells with the rCE18 were disrupted ultra-sonically, and the inclusion bodies were washed with a solution containing 0.2% deoxycholic acid sodium (DOC)and 2% DOC, respectively. Then they were denatured with 0.9% sodium lauroyl sarcosine (SKL) followed by dialysis and gel filtration to refold and purify the target protein. At the same time, this method was compared with GST-FF affinity chromatography and recovering from SDS-PAGE gel. Biological activity of purified rCE18 was analyzed with indirect ELISA, and the purity of the products was identified using SDS-PAGE. The purity of refolded inclusion bodies exceeded 60% and the total recovery of activated protein rCE18 was about 41.3%. The specificity of rCE18 reached up to 97.2% using indirect ELISA. An effective way for purifying and refolding rCE18 expressed in E. coli as inclusion bodies was established, rCE18 with higher purity and activity was obtained, which has the potential for developing diagnosis methods of porcine cysticercosis.
Animals
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Antigens, Helminth
;
biosynthesis
;
genetics
;
immunology
;
isolation & purification
;
Chromatography, Gel
;
Cysticercus
;
genetics
;
immunology
;
metabolism
;
Escherichia coli
;
genetics
;
metabolism
;
Inclusion Bodies
;
metabolism
;
Protein Renaturation
;
Recombinant Proteins
;
genetics
;
immunology
;
isolation & purification
10.Expression, purification and renaturation of Pol P51 antigen of HIV-1 strain CN54 and its application in antibody detection.
Jue HOU ; Jing SUN ; Zhiyong XU ; Wenling FAN ; Yixuan ZHANG ; Yong LIU ; Yanling HAO
Chinese Journal of Biotechnology 2010;26(2):201-206
To obtain the pure and soluble P51 antigen of HIV-1 strain CN54, we transformed the Escherichia. coli strain BL21 codonplus-RIL with recombinant plasmid pTHioHisA51 which carries a gene encoding the Polymerase (Pol) P51 antigen of HIV-1 CN54 formerly, and induced protein expression by IPTG. We purified the recombinant protein with Chelating Sepharose FF-Ni and DEAE-Sepharose FF column chromatography, then renatured the recombinant protein by dialyzation. Purified protein was identified by Western blotting. We labeled and coated antigen P51 in a dual-antigen sandwich system, and tested it with serum samples from HIV-infected individuals. The results showed that P51 was expressed as inclusion body, and represented about 50% of total cellular protein. After purification and renaturation, the purity of P51 was up to 95%. Western blotting and sandwich ELISA demonstrated that recombinant P51 had good anti-HIV antibody specificity and sensitivity. The results suggested that recombinant HIV-1 P51 can be prepared as diagnostic reagent, and provides valuable support for HIV-1 detection and vaccine research.
Escherichia coli
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genetics
;
metabolism
;
HIV Antibodies
;
blood
;
immunology
;
HIV Infections
;
immunology
;
virology
;
HIV Reverse Transcriptase
;
biosynthesis
;
genetics
;
immunology
;
HIV-1
;
classification
;
immunology
;
Humans
;
Protein Renaturation
;
Recombinant Proteins
;
biosynthesis
;
genetics
;
immunology
;
Sensitivity and Specificity