[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 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.
Jiang
Hanmin
H
College of Life Sciences, Nankai University, Tianjin 300071, China.
Chai
Xinjun
X
He
Bing
B
Zhao
Juan
J
Yu
Xinda
X
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D020932:Nerve Growth Factor / Q000096:biosynthesis* / Q000235:genetics; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C061397:pro-nerve growth factor, human; D020932:Nerve Growth Factor