Construction, expression and refolding of recombinant luteinizing hormone releasing hormone-angiogenin toxin
10.3760/cma.j.issn.1673-0860.2010.08.015
- VernacularTitle:人促黄体激素释放素-血管生成素重组毒素的构建表达及其复性的实验研究
- Author:
Zhi-Li NI
1
;
Qiu-Hang ZHANG
;
Qiu-Yi QU
;
Hai-Li L(U)
;
Shu-Ya FAN
;
Chao CAI
Author Information
1. 首都医科大学宣武医院
- Keywords:
Gonadotropin-releasing hormone;
Angiopoientins;
Immunotoxins;
Escherichia coli
- From:
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
2010;45(8):680-684
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express, purify and refold recombinant luteinizing hormone releasing hormone-angiogenin(LHRH-Ang) toxin using E. coli. expression system. Methods Recombinant LHRHAng expression vector was constructed by replacing of EGF fragment in plasmid pET28a/EGF-Ang with LHRH-P Ⅱ fragment amplified from plasmid pET28/MSH-PEA0. DNA sequencing would be used to verify the correction of fused LHRH-P Ⅱ -Ang gene. Then, E. coli strain BI21 (DE3) was transformed by pET28a/LHRH-Ang vector. Expression of recombinant LHRH-Ang toxin was induced by Isopropyl-β-D-Thiogalactoside( IPTG ). Refolding effects of gradient dialysis was evaluated by SDS-PAGE. Results Prokaryotic expression vector pET28a/LHRH-Ang, containing LHRH-P Ⅱ -Ang fusion gene, was constructed by PCR amplification, restriction enzyme digestion and ligation method. Sequence correction of fusion gene was confirmed by DNA sequencing After IPGT induction, recombinant LHRH-Ang protein was expressed in BL21 ( DE3 ) as inclusion body ,it took 18.43% of total protein. Inclusion body was resolved in 8 mol/L urea and purified by DEAE-Sepharose FF column, the purity was 85%. Recombinant LHRH-Ang toxin was refolded and concentrated by gradient dialysis and PEG 20000, respectively. Conclusions Recombinant LHRH-Ang protein was expressed in E. coli and refolded successfully.
