Study on the protein expression of CYP3A4 and CYP2C18

Abstract

Background As a drug enzyme, CYP450 has important significance in clinical treatment and the combined use of drugs, but the current research has not yet achieved high efficiency and large amount of expression in vitro. Therefore, the expression of CYP450 in vitro has become a hot spot.Objective Study the in vitro high-efficiency expression and time optimization of CYP3A4 and CYP2C18. Methods (1) CYP3A4 and CYP2C18 yeast recombinants were induced, and 24h, 48h and 72h bacterial liquid were collected respectively, to obtain the crude enzyme solution at different time periods, and then the enzyme solution at different time periods was subjected to SDS-PAGE protein electrophoresis to obtain its target bands. The results of comparative analysis between the protein bands and the Marker were observed by observing the gel running results. Finally, the enzyme activity was determined by liquid chromatography. Results The expressed proteins of CYP3A4 and CYP2C18 yeast recombinants were successfully induced. The target bands were obtained by SDS-PAGE protein electrophoresis and the optimal expression time was analyzed. Finally, the activity of the products was verified by liquid chromatography. Conclusion CYP3A4 and CYP2C18 yeast recombinants were successfully expressed in this experiment, and the most abundant expression of CYP3A4 and CYP2C18 was found at 72h by comparing the position of electrophoresis bands. At the same time, it was also verified that the expressed products were active, and CYP3A4 had the highest activity at 48h, which could be used for the study of CYP450 metabolism in vitr.