[Expression of the Mycobacterium tuberculosis cytochrome CYP51 gene in Escherichia coli].
Mycobacterium tuberculosis sterol 14 alpha-demethylase CYP51 is currently considered a promising target for a new generation of antitubercular drugs. Similarly to eucaryotic sterol 14 alpha-demethylases, candidate selective inhibitors of M. tuberculosis CYP51 belong to azoles, the N-substituted derivatives of imidazole and triazole. We have constructed a highly effective system for heterologous expression of MT CYP51 in E. coli. The recombinant protein was purified by metalloaffinity chromatography for primary in vitro screening of novel antitubercular azole compounds for their high affinity to the molecular target. Yield of the purified recombinant MT CYP51 was about 1.5 micromoles of the purified native protein per 1 l of E. coli cell culture. The recombinant protein interacted with ketoconazole with a Kd of 7.7 microM. MT CYP51 produced by our system for heterologous expression in E. coli may be used for initial testing of novel antimycobacterial azole drugs.
Shavkunov
A S
AS
Institute of Physico-Chemical Medicine, Malaya Pirogovskaya ul. 1a, Moscow 119828, Russia. a_s_shavkunov@hotbox.ru
Lazarev
V N
VN
Zgoda
V G
VG
Govorun
V M
VM
Levi
P
P
Janssen
P
P
Archakov
A I
AI
D000595:Amino Acid Sequence; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics*; D058886:Sterol 14-Demethylase
D017931:DNA Primers; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase