Studies On The Expression Of Rhine Chlamydomonas Reinhardtii Cytochrome P450 55B1 And Its Application In Nitric Oxide Detection

Rhine chlamydomonas cytochrome P450 55 B1(CYP55B1) is a kind of nitric oxide reductase. It can reduce ntric oxide to nitrous oxide with the electron supply with NADH. Nitric oxide(NO) exists in animals and plants widely. However, the concentration of NO is very low in vivo and it is liable to be oxidized, which brings a great challenge for the detection of NO. In this thesis we expressed CYP55B1 in heterologous system and studied the interaction of NO with CYP55B1 by spectroscopic method. We have developed a NO detection method by fluorescence spectroscopy based on CYP55B1. This thesis contains several parts as following: Firstly, according to the aimino acid sequence of CYP55B1 on NCBI website, it is identified that the protein is soluble with the molecular weight of 44 kD and isoelectric point of 6.74 by bioinformatics analysis. The tertiary structure model of native CYP55B1 was established and the model was assessed to be reliable by PROCHECK analysis.Secondly, the open reading frame of cyp55b1 gene was cloned to pPIC9 vector to construct the expression vector pPIC9-55b1. In order to express CYP55B1 in Pichia pastoris, different pH and different time was investigated. CYP55B1 was attained through secretion expression and intracellular expression. The secretion expression level of CYP55B1 is 41.84 nmmol/g after 48 h in pH 6 medium. The intracellular expression level of CYP55B1 is 166 nmmol/g after 36 h in p H6 medium. But the expression level was extremely low and unstable.Thirdly, vector pET28a-55b1 was constructed. Experiments were performed subsequently to express CYP55B1 in E.coli successfully. The expression level of CYP55B1 is 1597.6 nmol/g after 22 hours at 28 °C with addition of 1 mmol/L IPTG. Using His-tag column it was purified successfully with the concentration of 133.85 nmol/g. The molecular weight of CYP55B1 was confirmed to be between 45 kD and 66 kD by SDS-PAGE and western-blot, which is consistent with the result of bioinformatic analysis.Finally, the interaction between CYP55B1 and NO has been studied by fluorescence and UV-vis spectroscopic method. The results show that the interaction between NO and CYP55B1 leads to an increase of hydrophobicity in the miro-environment of tyrosine, while the interaction between NADH and CYP55B1 leads to an increase of hydrophobicity in the miro-environment of both tryptophan and tyrosine. The fluorescence intensity of iron porphyrin in CYP55B1 gradually increases with the addition of NO and regains the original value with the addition of NADH. When the concentration of NO below 18 ?mol/L, the linear relationship of fluorescence intensity and concentration of NO is as follows: F-F0 = 0.1456 CNO(?mol/L) + 0.0675. The detection of NO is slightly affected by certain interference factors such as CO?GSH?H2O2?KNO3?L-Arg?NaNO2 and ascorbic acid and it demonstrates the good selectivity of the method. And the dissociation constants, Kd, was calculated to be 7.742×10-5 mol/L through double-reciprocal plot, which indicates that NO has a strong affinity with CYP55B1. In contrast, NADH has little effect on the fluorescence intensity of iron porphyrin in CYP55B1. Furthermore, the catalytic mechanism of CYP55B1 to NO is verified by the spectroscopic method at the same time in this thesis.