Expression, purification and renaturation of proNGF in Escherichia coli / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 509-514, 2008.
Artigo
em Chinês
| WPRIM
| ID: wpr-276091
ABSTRACT
Nerve growth factor (NGF) promotes neuronal survival and differentiation and stimulates neurite outgrowth. NGF is synthesized as a precursor-proNGF in vivo. In this paper, a pET-proNGF prokaryocyte expression vector was constructed and transformed into E. coli BL21(DE3)pLysS. The proNGF was expressed in the form of non-active aggregated monomer in E. coli after induction with IPTG. SDS-PAGE revealed the proNGF expression product had a Mr.30.2 kD. Western blotting analysis showed that the protein had good antigenicity. Fusion protein was successfully purified by Ni2+-NTA affinity chromatography and cleaved by Enterokinase and 13.1 mg proNGF was obtained from 100 mL cell culture in a typical experiment. The protein was dialyzed in a redox system containing reduced and oxidized glutathione. RP-HPLC was used to analysis the result of the refolding. The refolded proNGF protein can induce neurite outgrowth of PC12 cells, which indicated that pro-form of NGF we obtained had biological activity.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Precursores de Proteínas
/
Proteínas Recombinantes de Fusão
/
Fator de Crescimento Neural
/
Renaturação Proteica
/
Escherichia coli
/
Vetores Genéticos
/
Genética
/
Metabolismo
Limite:
Humanos
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2008
Tipo de documento:
Artigo
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