Expression And Purification Of Recombinant Cytochrome P450nor And The Establishment Of Surface Plasmon Resonance Biosensor-based Immunoassays

Cytochrome P450 (P450) is a heme enzyme, whose CO complex gives Soret absorption at 450 nm in its optical absorption spectrum. It contains noncovalent-bound ferrin and has an electron delivery pathway by reversible change. After reduced by accepting electron and bound with CO, ferrins form optical absorption spectrum. The term "cytochrome P450" first appeared in literature in 1962. It was a microsomal membrane-bound hemoprotein without known physiological functions at that time and was characterized by a unique 450nm optical absorption peak of its carbon monoxide-bound form. Cytochrome P450 functions as the monooxygenase. P450s are involved in physiologically important processes including steroid metabolism, drug deactivation, procarcinogen activation, fatty acid metabolism, xenobiotic detoxification and wildly distributed in animal, plant and low eukaryotic organism. Cytochrome P450nor ( P450nor) is a unique cytochrome P450, which plays a key role in fungal denitrification. P450nor is a nitric oxide (NO) reductase (Nor) found in eukaryotic microorganisms, which reduces NO to nitrous oxide (N2 O) 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. The recombinant expression plasmids pRT-P450nor and pET-P450nor were constructed by inserting P450nor gene into the BamH I/ Hind III site of the prokaryotic expression vector pRSET and pET28, then they were transformed into E.coli BL21. In order to improve the expression level of the protein, the induced temperature and IPTG concentration were optimized. It indicated that optimal induced temperature was 30 C, whereas the change of IPTG concentration had little effect. The optimal induced conditions were as follows: the induced temperaturewas 30 C; The concentration of IPTG was Immol/L. Cytochrome P450nor was highly expressed by the inducement of IPTG. SDS-PAGE analysis showed that the molecular weight of the expressed protein was about 44KD. In this optimal condition, P450nor was expressed massively. The expressed product was then purified by DEAE-cellulose chromatography. The purified expressed protein showed one band in SDS-PAGE and the purification attained anticipative purpose.Surface plasmon resonance (SPR) biosensors have become an advance method of measuring biospecific interaction analysis (BIA) among the modern gene manipulation technique. Compared with conventional methods such as enzyme-linked immunosorbant assay (ELISA), it is a more convenient, sensitive and useful assay in real time. This new method plays an important role in the field of basic biological research, medical diagnosis and clinical therapy. Because of its high-sensitive and specific interaction between macromolecules, SPR biosensor is used for directly examining the reaction between targeted protein and corresponding antibody without careful purification. We introduced this new technology into our research in order to establish and optimize this advanced method with self-assembled SPR biosensor and hepatitis B surface antigen. The affected factors during measurement were discussed. It set a foundation for the research on the biospecific interaction of cytochrome P450nor and its antibody.