Expression of nerve growth factor from Agkistrodon halys Pallas in E. coli and its purification.
The nerve growth factor (NGF) gene of Agkistrodon halys Pallas was cloned into a secretive prokaryotic expression vector pET-22b+ which carried a C-terminal His. Tag sequence. After transforming into E. coli BL21 (DE3), NGF was induced to express at 30 degrees C by IPTG. SDS-PAGE analysis showed an induced expression product band which constituted about 20% of the total bacterial proteins. However, its molecular weight was larger than what was expected. Moreover, the analysis of product solubility revealed that NGF was in the form of inclusion bodies. The inclusion bodies were solubilized in 6 mumol/L guanidine HCl and purified directly by immobilized metal (Ni2+) chelation affinity chromatography. The product was renatured by dilution and air oxidation in the presence of 5 mumol/L CuSO4, and was proved active by examining the survival rate of PC12 cells in a serum-free medium.
Shanghai Institute of Biochemistry, Chinese Academy of Sciences, China.
L
L
Guo
X
X
Zhong
X
X
Wu
Y
Y
Zhou
D017836:Agkistrodon / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D004926:Escherichia coli; D020932:Nerve Growth Factor / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D020932:Nerve Growth Factor