Construction, expression and refolding of recombinant luteinizing hormone releasing hormone-angiogenin toxin / 中华耳鼻咽喉头颈外科杂志
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
;
(12): 680-684, 2010.
Artículo
en Chino
| WPRIM
| ID: wpr-276404
ABSTRACT
<p><b>OBJECTIVE</b>To express, purify and refold recombinant luteinizing hormone releasing hormone-angiogenin (LHRH-Ang) toxin using E. coli. expression system.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>Recombinant LHRH-Ang protein was expressed in E. coli and refolded successfully.</p>
Texto completo:
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Índice:
WPRIM (Pacífico Occidental)
Asunto principal:
Plásmidos
/
Ribonucleasa Pancreática
/
Proteínas Recombinantes de Fusión
/
Expresión Génica
/
Hormona Liberadora de Gonadotropina
/
Escherichia coli
/
Vectores Genéticos
/
Genética
/
Metabolismo
Idioma:
Chino
Revista:
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
Año:
2010
Tipo del documento:
Artículo
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