His(6) Fusion Expression of Myristoyl-CoA: Protein N-myristoyltransferase in E. coli and its Purification.
Saccharomyces cerevisiae Myristoyl-CoA: protein N-myristoyltransferase (NMT) gene was cloned into a His(6)-fusion expression vector pMFHT. After transforming into E. coli BL21 (DE3), His(6)-NMT was induced to express at 37 degrees by IPTG. SDS-PAGE analysis showed an induced expression product band of about 54 kD which constituted about 10% of the total bacterial proteins. The analysis of product solubility revealed that His(6)-NMT was predominantly soluble. On the basis of these results, His(6)-NMT was purified in one-step to 95% of purity from bacterial lysates using immobilized metal (Ni(2+)) chelation affinity chromatography. The in vitro labelling experiment demonstrated that His(6)-NMT had an activity similar to that of mature NMT, suggesting that the His(6)-tag did not affect the enzyme activity. His(6)-tag in the N-terminal of NMT makes it be possible to immobilized simply on Ni(2+)-IDA Sepharose 6B resin, which can be used to screen the peptide inhibitors of NMT from Phage Display random peptide library.
Xu
Zheng-Ping
ZP
Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China.
Duan
Zhi-Jun
ZJ
Chen
Chang-Zheng
CZ
Yang
Xin-Yin
XY
Li
Bo-Liang
BL