Expression, purification and renaturation of proNGF in Escherichia coli

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 expresse...

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Bibliographic Details
Published inShengwu gongcheng xuebao Vol. 24; no. 3; p. 509
Main Authors Jiang, Hanmin, Chai, Xinjun, He, Bing, Zhao, Juan, Yu, Xinda
Format Journal Article
LanguageChinese
Published China 01.03.2008
Subjects
Escherichia coli - genetics
Escherichia coli - metabolism
Genetic Vectors - genetics
Humans
Nerve Growth Factor - biosynthesis
Nerve Growth Factor - genetics
Protein Precursors - biosynthesis
Protein Precursors - genetics
Protein Renaturation
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - isolation & purification
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Summary: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.
ISSN:1000-3061