[Construction, expression and refolding of recombinant luteinizing hormone releasing hormone-angiogenin toxin].
OBJECTIVE
To express, purify and refold recombinant luteinizing hormone releasing hormone-angiogenin (LHRH-Ang) toxin using E. coli. expression system.


METHODS
Recombinant 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.


RESULTS
Prokaryotic 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.


CONCLUSIONS
Recombinant LHRH-Ang protein was expressed in E. coli and refolded successfully.
Department of Otorhinolaryngology Head and Neck Surgery, Capital University of Medical Science, Beijing 100053, China. nzl55@sina.com
Zhi-li
ZL
Ni
Qiu-hang
QH
Zhang
Qiu-yi
QY
Qu
Hai-li
HL
Lü
Shu-ya
SY
Fan
Chao
C
Cai
D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D007987:Gonadotropin-Releasing Hormone / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012259:Ribonuclease, Pancreatic / Q000096:biosynthesis* / Q000235:genetics
D011993:Recombinant Fusion Proteins; D007987:Gonadotropin-Releasing Hormone; C046366:angiogenin; D012259:Ribonuclease, Pancreatic