Expression, purification and renaturation of proNGF in Escherichia coli.
- Author:
Hanmin JIANG
1
;
Xinjun CHAI
;
Bing HE
;
Juan ZHAO
;
Xinda YU
Author Information
1. College of Life Sciences, Nankai University, Tianjin 300071, China.
- Publication Type:Journal Article
- MeSH:
Escherichia coli;
genetics;
metabolism;
Genetic Vectors;
genetics;
Humans;
Nerve Growth Factor;
biosynthesis;
genetics;
Protein Precursors;
biosynthesis;
genetics;
Protein Renaturation;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
isolation & purification
- From:
Chinese Journal of Biotechnology
2008;24(3):509-514
- CountryChina
- Language:Chinese
-
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.
