Quinone Oxidoreductase 1(NQO1) In Different Insect Cells Expression Conditions Optimization

Quinone oxidoreductase 1(NQO1)is a widely existed reductase.As a multifunctional protein,it plays many different roles in the organism.Such as stable oxidation balance,detoxification,prevent cell apoptosis and so on.The role of quinone oxidoreductase 1(NQO1)in cancer has been demonstrated in various studies,and elevated levels of NQO1 have been detected in many different types of cancer cells,such as breast,ovarian,liver,esophageal,and pancreatic cancers.Therefore,inhibition of NQO1 is considered as a potential treatment for cancer.In this paper,the exogenous expression conditions of the enzyme were screened,and the protein with higher expression level was obtained after purification,thus laying a foundation for the analysis of its structure and function.In this study,by screening the expression conditions of quinone oxidoreductase 1(NQO1)in insect cell expression system,the expression conditions of NQO1 were optimized from three common conditions involved in the expression process of different insect cells,different viral amounts and different expression time points,so as to achieve a high yield of target protein.This study used the In-Fusion Cloning technology will build NQO1 gene on p Fast Bac1 carrier,sequencing right after the transformed cells,recombinant baculovirus extracted plasmid,the recombinant baculovirus plasmid transfection Sf9 cells,the virus get P0 generation,continue to get P1 generation of virus infection,in turn,drops with high degrees of P2 is prepared for expressing screening of the virus.Three commonly used insect cells,Sf9,Sf21 and Hi-5,were inoculated with MOI1,MOI5 and MOI10.After inoculated with the virus at 24 h,48h,72 h and 96 h,the cells were harvested and identified by SDS gel electrophoresis after a small amount of affinity purification.Under these optimized conditions,a large amount of 400 m L was cultured and purified by AKAT system,and then the expression was determined by high performance liquid chromatography(HPLC)and liquid chromatography-mass spectrometry(LC-MS).The results showed that the optimal expression conditions of quinone oxidoreductase 1(NQO1)in insect cell expression system were Sf21 cells,MOI1,expressed 48 h,and purified with 400 ml of quinone oxidoreductase 1 was 1.17 mg.Finally,HPLC and LC-MS were used to identify the expression of the protein.This study laid a foundation for the analysis of its structure and function,and also laid a foundation for the development of targeted small molecule new drugs based on this enzyme.