Expression Of Human Cytochrome P4502C9and Fusarium Oxysporum Cytochrome P45055A1in Pichia Pastoris

Cytochrome P450(CytochromeP450, CYP) are a class of hemoglobinsuperfamily that can be combined with CO resulting in maximum absorption peak at450nm. It is widely distributed in nature and a variety of animals, plants and a largenumber of microorganisms have been found CYPs’ abound. Cytochrome P450is animportant part of multifunctional enzyme, with a broad and functional diversityof substrates that can metabolize a variety of endogenous and exogenoussubstances.It’s application is related to medicine, agriculture and environment.Pichia pastoris (P.pastoris) is a kind of a eukaryotic expression system with astrong alcohol oxidase (Alcohol Oxidase, AOX) promoter. Post-translationalmodification process to exogenous protein forms active protein. And the secretion ofheterologous protein simplifies the protein purification.In this paper, the Pichia expression system has been applied to express two P450genes. It mainly contains：(1)The genes of human cytochrome p4502c9and Fusariumoxysporum cytochrome p45055a1are homologously integrated into the yeast genomeso that it can copy stably. We construct cyp2c9and cyp55a11gene into the yeastexpression vector, then large-scale amplificate and linear the recombinant plasmidwith restriction enzyme after gene sequencing. Purified the linearized plasmid andelectroporation transformed into yeast GS115competent cells, so that the foreigngene integrated into the yeast cells.(2) The positive strains reorganized have been screened by using of MD platesand PCR. The transformants were picked from MD plates and genome of positivestrains was extracted for PCR. There are two bright bands in the pPIC3.5K-2c9positive strains respectively. One is about2200bp (aox1gene) and another is1700bp(including the target gene which are1500bp and vector homologous sequences areapproximately214bp); there are two bright bands in the pPIC9-55a1positive strains respectively. One is about2200bp (aox1gene) and another is1700bp (including thetarget gene which are1200bp and vector homologous sequences are approximately490bp). The types of recombinants by the initial screening of the nutrient medium andPCR have been found to facilitate the next expression.(3) SDS-PAGE electrophoresis and Western Blotting have been used to validatethe expression of the recombinant strains. Culture His+Mut+strain with BMGYmedium first and then resuspend sediment with BMMY medium after centrifagationto induce expression. Add methanol to final concentration of0.5%every24hours andcollect samples until96hours. Place samples of each point in time at lowtemperatures for sonication treatment and then electrophoresis analysis after beingconcentrated. SDS-PAGE shows that there is no particularly obvious purpose band.Western Blot shows that the specific protein bands are in the expected location,indicating the protein expression level is not high. But the conclusion can beconfirmed that CYP2C9and CYP55A1have been expressed in Pichia pastoris.