Cloning and Expression of the Yeast Saccharomyces cerevisiae MyristoyL-CoA: Protein N-Myristoyltransferase Gene.
The gene encoding the myristoyl-CoA: protein N-myristoyltransferase from yeast S. cerevisiae (YSCNMT) has been amplified with the synthetic oligonucleotides by PCR, cloned into the pBluescript KS+ vector and analyzed by DNA sequencing. Then, an expression plasmid (pMFT7-5-NMT) containing the cloned YSCNMT gene controlled by T7 promoter has been constructed and transformed into E. coli BL21(DE3). Analyzed by SDS-PAGE, an IPTG-induced product with the same molecular size (53 kD) as that deduced from the known YSCNMT amino acid sequence was observed. It accounted for about 39% of the total cellular proteins, the soluble form of which accounted for about 34% of the total soluble cellular proteins and was purified to homogeneity in a single step using P11 phosphocellulose chromatography. The N-terminal amino acid sequencing revealed that the expressed protein began with the sequence encoded by the cloned YSCNMT gene and did not contain the N-terminal methionine. It was also observed that the expressed product had apparent YSCNMT activity in the in vitro assay.
Duan
Zhi-Jun
ZJ
Shanghai Institute of Biochemistry, Academia Sinica, Shanghai 200031, China.
Chen
Chang-Zheng
CZ
Li
Bo-Liang
BL