Expression Purification and Activity ofhuman Myristoyl-CoA:N-myristoyltransferase.
A gene encodinghuman myristoyl-CoA: protein N-myristoyltransferase (hNMT) from a brain cDNA libraryhas been obtained by PCR amplification and DNA sequencing. Then the mature-type and His(6)-fusion-type expression plasmids (pMF-hNMT(3) and pMFHT-hNMT(2)) containing the hNMT gene under control of T7 promoter have been constructed and transformed into E. coli BL21(DE3). SDS-PAGE analysis showed that the recombinant hNMT products expressed at 37 degrees were almost unsoluble but most of the His(6)-hNMT product expressed at lower temperature (22 degrees ) was soluble and its yield was about 7% of the total soluble cellular proteins. By immobilized metal (Ni(2+)) chelation affinity chromatography up to 80% His(6)-hNMT was purified by one step from bacterial lysate. The labelling experiment in vitro domonstrated that the expressed and purified His(6)-hNMT had an obvious catalytic activity to transferring myristoyl group.
Shanghai Institute of Biochemistry the Chinese Academy of Sciences Shanghai 200031, China. boliang@server.shcnc.ac.cn
Xin-Ying
XY
Yang
Lan
L
Chen
Hu
H
Chen
Zheng-Ping
ZP
Xu
Bo-Liang
BL
Li