【作者】 陈舒丽；

【导师】 刘德立；

【作者基本信息】 华中师范大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 细胞色素P450是一类能与CO结合，形成的复合物在450nm附近具有最大吸收峰的血红蛋白的总称。它含有非共价结合的血红素铁，并在细胞内通过可逆性的变化而进行电子传递。当血红素铁接受电子被还原后再与CO结合，将产生细胞色素P450的特征光谱。细胞色素P450种类繁多，功能多样，是一个庞大的超基因家族。它的化学本质是蛋白质，分子质量在43-60kD之间。细胞色素P450具有单加氧酶的作用，并能参与机体解毒、甾体激素的合成、脂肪酸代谢等重要的生理反应，并广泛分布于动物、植物和微生物中。细胞色素P450nor是一类独特的细胞色素P450，它在真菌反硝化中扮演着重要角色。P450nor起着NO还原酶的作用，能以NAD（P）H为直接电子供体将NO还原成N₂O。虽然它属于P450超家族，但没有单加氧酶活性。迄今为止，已从真菌和酵母中克隆了多种细胞色素P450nor基因，表明其存在普遍性。本研究改造了原有的未能高效表达的重组表达载体pRSET-p450nor，通过PCR方法，去除了P450nor起始密码子ATG上游的59个碱基序列，将真菌细胞色素P450nor基因首先克隆至克隆载体pBlue上，得到重组载体pBlue—p450nor。然后亚克隆到高效表达载体pET-28和穿梭表达载体pAUR123上，得到了重组表达载体pET-p450nor和重组穿梭表达载体pAUR123-p450nor。将重组表达载体pET-p450nor转化大肠杆菌BL21菌株，该基因在30℃、0.1mM IPTG诱导下获得高效表达融合蛋白His-P450nor。SDS-PAGE蛋白电泳表明在45kD处出现强特异性带。将大量表达的重组蛋白用Ni-NTA亲和层析柱纯化，得到了单一的目的条带。将重组穿梭表达载体pAUR123-p450nor首先转化大肠杆菌BL21，经筛选鉴定后转化酿酒酵母AH22。在YEPE培养基上培养带有P450nor基因的酿酒酵母，30℃，培养36h使目的蛋白表达，SDS-PAGE电泳表明在45kD处出现表达条带。本研究还探讨了温度、IPTG浓度对重组蛋白在原核表达中的影响。结果表明：在20℃以下，不易形成包涵体，但蛋白表达量较小；在1mM的IPTG浓度和0.1mM的IPTG浓度的诱导下，蛋白表达量并无明显差异。培养基成分的不同对目的蛋白在真核中的表达也有影响，蛋白的表达量在YEPE培养基比在YEPD培养基中大。更多还原

【Abstract】 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.更多还原