Cloning, Expression, And Purification Of The Recombinant Cytochrome P450nor In Escherichia Coli And Saccharomyces Cerevisiae

Cytochrome P450 (P450) is a heme enzyme, whose complex gives Soret absorption at 450nm in its optical absorption spectrum. It contains noncovalent-bound ferrin and has an electron delivery pathway by reversible optical absorption spectrum. These proteins are not, in fact, "cytochromes" in the true sense of word and the term "heme-thiolate protein"Was preferred instead. Its molecular weight is between 43kD to 60kD. It is well known that P450 are widely distribute in almost all eukaryotes, prokaryotes, even in hyperthermophilic archaea and catalyzing not only monooxygenation, but also many other biological reaction .Therefore, the physiological function of P450s are extremely important including steroid metabolism, drug deactivation, procarcinogen activation, fatty acid metabolism, xenobiotic detoxification and catabolism of various exogenous compounds as source of energy.Cytochrome P450nor (P450nor) is a unique cytochrom P450, which plays a key role in fungal denitrification. Cytochrome P450nor is a nitric oxide (NO) reductase (Nor) found in eukaryotic microorganisms, which reduces NO to nitrous oxide (N₂O) by directly accepting electrons from NADH or NADPH. Unlike other P450s, P450nor has no monooxygenase activity although it belongs to the P450 super family (CYP55A). Several P450nor genes have been cloned from fungi and yeast, indicating a wide distribution of P450nor in low eukaryotes.Recombinant plasmid pBlue-p450nor was constructed by inserting P450nor gene into cloning vector pBluescript II. P450nor gene was amplified by PCR method using pRSET-p450nor as template which was constructed before. 59 bp of the upstream sequence of P450nor gene had been deleted. The recombinant expression plasmids pET-p450nor was constructed by subcloning P450nor gene into the prokaryotic expression vector pET-28, then they were transformed into Escherichia coli BL21.The vector allows overproduction and single-step purification of His₆-tagged cytochrome P450nor by the facilitation of metal (Ni²⁺) chelate affinity chromatography. The expression level of cytochrome P450nor was high at 30℃after 0.1mM IPTG inducing. SDS-PAGE analysis showed a specific band (about 45kD), indicating the target protein expressed successfully. The overexpressed cytochrome P450nor was purified to electrophoretic homogeneity by the facilitation of metal (Ni²⁺) chelate affinity chromatography. The recombinant E.coli- S. cerevisiae shuttle vector pAUR123-p450nor was constructed based on plasmids pBlue-P450nor and pAUR123. The P450nor gene, got from pBlue-p450nor, was inserting to pAUR123 to construct pAUR123-P450nor. Then pAUR123-p450nor was transformed into E. coli BL21 for identification and then transformed into S. cerevisiae AH22. The positive recombinant S. cerevisiae AH22 harboring with P450nor gene, were cultured at 30℃on YEPE medium for 36 hours. SDS-PAGE was used to verify the product of expressed P450nor gene, and the results indicated that cytochrome P450nor was expressed in S.cerevisiae AH22, a specific band (about 45kD) was deteced.Other influence factors are also detected, such as temperature and IPTG concentration, on the expression level of P450nor when expressed in E. coli BL21. It indicated that, it was not easy to form inclusion body below 20℃, but the expression level was low. There is no obvious difference on expression level of P450nor between 1mM IPTG and 0.1mM IPTG. When S. cerevisiae AH22 with cytochrome P450nor gene was cultured on YEPE medium and YEPD medium at the same time, we detect more P450nor protein on YEPE medium than on YEPD medium.