Expression, Purification
and Activity ofhuman Myristoyl-CoA:N-myristoyltransferase
YANG Xin-Ying, LI Bo-Liang
( Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai
200031, China )
CHEN Lan
( Shanghai Institute of Materia Medica, the Chinese Academy of Sciences, Shanghai
200031, China )
CHEN Hu, XU Zheng-Ping
( Department of Biological Sciences and Technology, Zhejiang University, Hangzhou
310027, China )
Abstract 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 His6-fusion-type expression plasmids (pMF-hNMT3
and pMFHT-hNMT2) 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 ºC were almost unsoluble, but most of the His6-hNMT
product expressed at lower temperature (22 ºC) was soluble and its yield was
about 7% of the total soluble cellular proteins. By immobilized metal (Ni2+)
chelation affinity chromatography, up to 80% His6-hNMT was purified
by one step from bacterial lysate. The labelling experiment in vitro
domonstrated that the expressed and purified His6-hNMT had an
obvious catalytic activity to transferring myristoyl group.
Key Words Human NMT; PCR; gene
expression; His6 fusion expression; metal (Ni2+)
chelation affinity chromatography
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