[Expression, purification and renaturation of proNGF in Escherichia coli].
Sheng Wu Gong Cheng Xue Bao
; 24(3): 509-14, 2008 Mar.
Article
em Zh
| MEDLINE
| ID: mdl-18589832
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Precursores de Proteínas
/
Proteínas Recombinantes de Fusão
/
Fator de Crescimento Neural
/
Escherichia coli
Limite:
Humans
Idioma:
Zh
Revista:
Sheng Wu Gong Cheng Xue Bao
Assunto da revista:
BIOTECNOLOGIA
Ano de publicação:
2008
Tipo de documento:
Article
País de afiliação:
China