Construction, expression and refolding of recombinant luteinizing hormone releasing hormone-angiogenin toxin.
- Author:
Zhi-li NI
1
;
Qiu-hang ZHANG
;
Qiu-yi QU
;
Hai-li LÜ
;
Shu-ya FAN
;
Chao CAI
Author Information
- Publication Type:Journal Article
- MeSH: Escherichia coli; metabolism; Gene Expression; Genetic Vectors; Gonadotropin-Releasing Hormone; biosynthesis; genetics; Plasmids; Recombinant Fusion Proteins; biosynthesis; genetics; Ribonuclease, Pancreatic; biosynthesis; genetics
- From: Chinese Journal of Otorhinolaryngology Head and Neck Surgery 2010;45(8):680-684
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo express, purify and refold recombinant luteinizing hormone releasing hormone-angiogenin (LHRH-Ang) toxin using E. coli. expression system.
METHODSRecombinant LHRH-Ang expression vector was constructed by replacing of EGF fragment in plasmid pET28a/EGF-Ang with LHRH-PII fragment amplified from plasmid pET28/MSH-PE40. DNA sequencing would be used to verify the correction of fused LHRH-PII-Ang gene. Then, E. coli strain BL21 (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.
RESULTSProkaryotic expression vector pET28a/LHRH-Ang, containing LHRH-PII-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.
CONCLUSIONSRecombinant LHRH-Ang protein was expressed in E. coli and refolded successfully.
