Prokaryotic expression and functional analysis of molecular chaperone Acr2 protein of Mycobacterium tuberculosis / 中国生物制品学杂志

@#Objective To express the molecular chaperone Acr2 protein of Mycobacterium tuberculosis(Mtb)in E.coli and analyze the function. Methods The recombinant plasmid pET-28a-Acr2 was transformed into competent E. coli BL21(DE3),and induced by IPTG. The expressed His-Acr2 protein was purified by Ni-NTA chromatography and SuperdexTM200 10/300 GL gel filtration chromatography to obtain Acr2 protein. The Acr2 protein was refolded by spontaneous refolding and reassembly after thermal denaturation(100 ℃ for 15 min)and chemical denaturation(8 mol/L urea,37 ℃ for 4 h).The secondary structure of Acr2 protein before and after denaturation-renaturation was detected by circular dichroism spectroscopy and non-denaturing SDS-PAGE,and the molecular chaperone function of Acr2 protein in vitro was detected by substrate binding assay. Results The purified Acr2 protein had the relative molecular mass of about 232 000,the purity of over 90%,and the concentration of about 2 mg/mL,which recovered its natural secondary structure after denaturationrenaturation,and formed stable complexes with the denatured malate dehydrogenase(MDH)at 48 ℃. Conclusion The Acr2protein can restore its natural molecular conformation with molecular chaperone activity in vitro after denaturation-renaturation treatment,providing a new strategy for the preparation of Mtb protein antigen with natural activity.

Expression,purification and renaturation of recombinant human collagen-binding bone morphogenetic protein-2 from Escherichia coli / 吉林大学学报(医学版)

Objective:To construct the Escherichia coli (E. coli)expression system for preparation of the bone morphogenetic protein-2 (BMP2)with collagen-binding domain (CBD),and to study the methods and conditions for expression, purification and renaturation of CBD-BMP2.Methods:CBD sequence was cloned into the N-terminal of BMP2 sequence, the recombinant vector pet21b/CBD-BMP2 was constructed and transformed into E.coli BL21.The expression of recombinant protein was induced using isopropylβ-D-thiogalactopyranoside (IPTG) at 37 ℃.Ni-NTA chelate chromatography was used to purify CBD-BMP-2.Denaturing CBD-BMP2 was refolded by dilution method using ultrapure water.The refolding CBD-BMP2 was filtered through a 0.22μm microfiltration membrane for degermation.The recovery rate was calculated by the ratio of the protein concentration before and after degermation. The expression, purification, and renaturation of recombinant protein were detected by SDS-PAGE method.The concentration of CBD-BMP2 was detected by BCA assay.Results:The recombinant vector pet21b/CBD-BMP2 was successfully transformed into E.coli BL21,and the recombinant protein was expressed as inclusion bodies in E.coli.The SDS-PAGE results showed denaturing protein was dissolved in supernatant of lysis buffer with 8 mol·L-1 urea and the purified recombinant protein existed in elution buffer B with relative molecular mass about 14 000.Two bands (14 000 and 28 000)were seen in the SDS-PAGE picture,which indicated that the monomer was successfully refolded into dimer by dilution method.The concentrations of recombinant protein before and after degermation were 110 and 80 mg · L-1 , respectively, and the recovery rate was about 73%. Conclusion:The recombinant vector pet21b/CBD-BMP2 is transformed into E.coli BL21 successfully,and the recombinant CBD-BMP2 is expressed and refolded efficiently. The methods of prokaryotic expression system for preparing recombinant CBD-BMP2 protein are established.

Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

Characterization of denaturation and renaturation of DNA for DNA hybridization

OBJECTIVES: The present study was designed to systematically characterize the denaturation and the renaturation of double stranded DNA (dsDNA), which is suitable for DNA hybridization. METHODS: A series of physical and chemical denaturation methods were implemented on well-defined 86-bp dsDNA fragment. The degree of each denaturation was measured and the most suitable denaturation method was determined. DNA renaturation tendency was also investigated for the suggested denaturation method. RESULTS: Heating, beads mill, and sonication bath did not show any denaturation for 30 minutes. However probe sonication fully denatured DNA in 5 minutes. 1 mol/L sodium hydroxide (alkaline treatment) and 60% dimethyl sulfoxide (DMSO) treatment fully denatured DNA in 2-5 minutes. CONCLUSIONS: Among all the physical methods applied, the direct probe sonication was the most effective way to denature the DNA fragments. Among chemical methods, 60% DMSO was the most adequate denaturation method since it does not cause full renaturation during DNA hybridization.

Expression and activity identification of recombinant human stanniocalcin 1 / 军事医学

Objective To obtain recombinant human stanniocalcin 1 ( STC1 ) with biological activity in Escheri.coli cells expression.Methods The gene was cloned into pET32b( +) vector by fused with thioredoxin and His tag .E.coli BL21(DE3) competent cells were transfomed by the recombinant vector .After renaturation, the fusion protein was digested with thrombin and intact STC1 protein was purified from the digested protein using Ni ion affinity chromatography .Recombi-nant humanSTC1 protein was confirmed by Western blot analysis using goat anti-STC1 antibody.The biological activity of STC1 in rat was assayed using standard method for assessment of renal function .Results The recombinant human STC 1 fu-sion protein is successfully expressed in Escherichia coli, the fusion protein was purified by affinity chromatography from the inclusion body and renaturated .Intact hSTC1 protein was released by thrombin digestion and purified by Ni ion affinity col-umn.The intact STC1 proteins was confirmed by Western blot analysis .Rat bioassay revealed that STC1 boosted phosphate reabsorption.Conclusion Recombinant STC1 protein was successfully expressed and has native biological activities .This protein could be used as an antigen for the preparation of monoclonal antibody against humanSTC 1.

Reteplase fusion protein：expression, purification and the effect of chaperones on its renaturation / 安徽医科大学学报

Objective To construct the prokaryotic expression system of reteplase fusion protein and research the effect of chaperones on its renaturation. Methods Inserted the reteplase gene into the prokaryotie expression vector PET32a and then expressed it by the induction of IPTG in E. coli BL21. Researched the effect of chaperones on the renaturation of fusion protein by adding different chaperones. Results The analysis of SDS-PAGE and Western blot indicated that reteplase fusion protein was expressed correctly. Chaperones DsbA,pKJE7,pTf16 had the conspicu-ous effect on the renaturation of fusion protein. The result of activity assay indicated that the refolded reteplase fu-sion protein had fibrinolytic activity. Conclusion Chaperones can promote renaturation of reteplase fusion protein.

Study on expression,purification and biological characteristics of recombinant human interferon-epsilon in E.coli / 中华微生物学和免疫学杂志

Objective To construct a novel recombinant human IFN-ε,and to analyze its physi-cal,chemical properties and biological characteristics.Methods Human genomic DNA was used as the template to synthesize IFN-ε gene by PCR.The sequence was cloned into plasmid vector pET-32a(+),and the recombinant plasmid pET-32a(+)/IFN-ε was transformed into E.coli BL2l(DE3).The form of ex-pression product was inclusion bodies.After purification and renaturation.high purity active protein IFN-εwas achieved.The final product was tested for its physical.chemical properties and biological characteristics including anti-viral and anti-proliferative acfivities.Results IFN-ε was expressed in inclusion body in E.coli.After the protein renaturation and purification,the purity was more than 95%.The rhIFN-ε protein had a specific anti-viral activity of about 1.2×103 IU/mg.Its anti-poliferative activity is obvious and can in-duce cells to produce anti-viral protein MxA.Conclusion Human IFN-ε protein was expressed successful-ly.and this protein has anti-virus and anti-prolireration activity.

Strong Expression of Recombinant Human Morphogenetic Protein-4 in Escherichia coli and its Bioassay in vivo / 中国生物工程杂志

Objective:To produce rhBMP-4 with bioactivity in E.coli. Methods: The full-length human BMP-4 gene was mutated by PCR without changes in amino acid sequence, then the synthesized gene was cloned into plasmid pET-3c, transducted into BL21(DE)plysS, and induced by adding IPTG to a final concentration of 1.0 mmol/L. The protein product was purified using ion-exchange chromatography method and then renaturated, bioactivity was checked by C2C12 differentiation in vitro and mouse ectopic bone formation in vivo. Results: A 438 bp gene fragment encoding mature peptide of hBMP-4 was cloned , the protein product was mostly in the form of inclusion body, after renaturation, the engineering protein shows better bioactivity. Conclusion:The mutant strategy can enhance the expression of bioactive rhBMP-4 in E.coli expression system.

The Renaturation and Activity Study of LexA From Pseudomonas aeruginosa / 中国生物工程杂志

Objective:To optimize the renaturation procedure of denatured LexA,prepare the repressor LexA from Pseudomonas aeruginosa(PA),which have the satisfactory biologic activity.Methods:The LexA was renatured by the GSH/GSSG dilution method,and the renatured protein were purified by Ni2+ chelate affinity chromatography and gel filtration chromatography,following desalination by Sephadex G25 gel column.The renaturation result were detected by the native polyacrylamide gel electrophoresis and RPHPLC.The immunological activity of all LexA proteins,including the denatured,renatured protein and the renatured protein that was treated with the DTT,were determined by Western blot.Results:The renatured LexA appears both monomer and multimer,which is confirmed by the native polyacrylamide gel electrophoresis analysis and RPHPLC.Gel retardation experiments shows that the renatured LexA have satisfactory biologic activity.

Prokaryotic Expression, Purification and Characterization of Recombinant Human Interleukin-17 / 中国肿瘤生物治疗杂志

Objective: To select suitable conditions for prokaryotic expression and purification of rhIL-17. Methods: rhIL-17 was expressed in E. coli host under heat induction. After compared among the expression amounts in different media under different heat induction time, the most suitable conditions was selected. The target protein was present in the form of inclusion body. The precipitate of inclusion was obtained and purified after 6M guanidine solublization or 2% SDS solublization. Results: Either protocol could yield rhIL-17 with high purity and stable activity. The SDS solublization mehthod gives rise to much more higher productivity than the guanidine solublization method. Conclusion: rhIL-17 were expression in E. coli system and purified to homogenicity by SDS solublization methods with high productivity.

Study on Purification and Renaturation of His6-?TNF?Fusion Protein (IB) / 中国肿瘤生物治疗杂志

The E. coli BL21(DE3) strain bearing the plasmid(T_7lac/His6-tag) with the h?TNF?recombinant gene was grown in LB medium containing SO?g/ml kanamycin, at 37 ℃ , up to the late logarithmic phase(OD590 ~ 0.5)then induced with IPTG (final concentration lmmol/L)for 5 hours. SDS-PAGE analysis revealed that expression level of the product(His6-?TNF?was up to 45% of the total bacterial proteins and was mainly as insoluble inclusion bodies ( IBs) . After cell lysis, the IBs was separated by centrifugation, dissolved in 7mol/L urea, then purified by Ni column ( Ni~(2+) -Sepharose 6B) . And the purity of more than 95% and the recovery of about 90% were obtained. The purified product was refolded with the renaturation buffer under low temperafure(

Purification,renaturation and antiviral effects of recombinant cyanovirin-N on herpes simplex virus type 1 / 中国海洋药物

Objective To purify,renature and explore the antiviral effects of recombinant cyanovirin-N(CV-N)on herpes simplex virus type 1(HSV-1).Methods The recombinant CV-N was purified with Ni Sepharose column and renatured by dilution method.The antiviral activities of CV-N were carried out in Vero cells by observing cytopathic effect(CPE)and by using MTT colorimetric assay for vital cell rate.Results SDS-PAGE showed that the purified protein was in the position of 11KDa with only one clear band.The renatured CV-N had little cytotoxic effect on Vero cells,it could not directly inactivate HSV-1 infectivity.CV-N not only interfered in adsorption of HSV-1 to Vero cells but also inhibited HSV-1 biosynthesis in the cells,which were more effective than the positive control Acyclovir.Conclusion CV-N exhibited pronounced antiviral activities agaist HSV-1,further development of CV-N might yield novel candidates of antiviral drugs.

Over-expression of Proapolipoprotein AI_(Milano) in Escherichia coli / 微生物学通报

To facilitate the mutation of Milano site, the whole proapo AI_(M) gene was divided to two fragments to synthesize by RT-PCR. The optimization of upstream gene sequence was carried out to improve proapo AI_(M) expression level in E. coli. After induction with a shift of temperature, yields of recombinant proapo AI_(M) achieved about 45 % of total cell protein and the recombinant proapo AI_(M) was expressed as a form of inclusion body in cells. The renaturation of protein was carried with a hydrophobic interaction column,the results showed that recombinant proapo AI_(M) had similar functional properties identical to those of native protein.