Exploring The Pichia Pastoris-based Bioreactor:Establishment Of The Expression System For Human Cytochromes P450

Human Cytochrome P450s involved in drug metabolism are important phase I enzymes in our body. These indicators including of absorption, distribution, metabolism, excretion and toxicity (ADME/Tox) are very important for the ultimate success of drug candidates in the process of research and development. The FDA regulations for new drugs development require that the concentration of a variety of drug metabolites must be determined when the metabolites from the drugs metabolized by P450enzymes are toxic and have impacts on the target tissues or the body organizations. Toxicity of the metabolites needs to be analyzed if the amounts are greater than10%of the overall drugs. The milligrams or even less of synthesized metabolites are usually used in the toxicity tests. It becomes complicated and time-consuming for such small amounts of the metabolites to be synthesized and separated using traditional chemistry. It also effects the sequential qualification and quantification of the metabolites.The research of drug metabolism has being carried out from the traditional animals’experiments into the stage of studies in vitro gradually. The change is due to the development of molecular biology and genetic engineering on a great extent. We would like to express the recombinant CYP450s highly using the heterologous expression system so as to produce metabolites. Then, we will establish the metabolic screening system in vitro so that we can forecast of the level of metabolism in vivo by metabolism data in vitro. Many European and American countries will have such a study during the new drug declaration. Yeast expression system is being widely used because of its efficient and economical.CYP450s, known as drug metabolizing enzymes, involved in the chemical transformation of many endogenous and exogenous compounds in human body and metabolic processes of the most drugs. CYP3A subfamily is an extremely important part of the whole enzymes and is about30%of the total CYP contents. About50%of drug metabolism involved in CYP450is related to the CYP3A. CYP3A5is once considered to pseudogenes lacking of function. But CYP3A5has gradually become a new hotspot in recent years and it is a blank that express CYP3A5to produce metabolites using yeast.CYP3A5is an important member of the3A family, the full gene located on human chromosome7is1726bp and cDNA length is1509bp. Many drugs on the market are metabolized by CYP3A. Pichia pastoris is used to express CYP4503A5and we hope to establish yeast system to produce metabolites standards in our study. The following work is:Firstly, CYP3A5gene is homologously integrated into the yeast genome so that it can copy stably. We sent recombinant plasmid to sequence after CYP3A5gene is constructed into the yeast expression vector-Ppic3.5k. Conversion of the E. coli DH5a and then linear the recombinant plasmid with restriction enzyme named Sall. We use ethanol to deposit the linearized plasmid and recovery it. Mix pastoris GS115competent cells and the linearized plasmid and then shocks to them by electric under the conditions of voltage1.5kV, capacitance25μF, resistance of200O. Thus CYP3A5gene is integrated into yeast cells by electroporation and will be integrated into the yeast genome by homologous recombination.Secondly, we screen the the positive strains reorganized by using of MD plates and PCR. Immediately add ice-cold sorbitol to the mixture after electroporation and place it in30℃for an hour. Then transfer the mixture to MD plates. Extract genome of positive strains for PCR reaction after3days. Wild-type strain appear2.1kb bands (AOX1gene) and there are two bright bands in the positive strains respectively. One is about2.1kb (AOX1gene) and another is1.73kb (including the target gene which are1509bp and vector homologous sequences are approximately220bp). That we find the types of recombinants by the initial screening of the nutrient medium and PCR facilitates the next expression.Thirdly, using SDS-PAGE protein electrophoresis and Western Blotting to validate the expression of the recombinant strains. Culture His+Mut+strain with BMGY medium first and then resuspend sediment with BMMY medium (1%methanol) after centrifugation to induce expression. Add methanol to final concentration of1%every24hours and collect samples until96hours. Place samples of each point in time at low temperatures for sonication treatment and then electrophoresis analysis after being concentrated. SDS-PAGE shows that there is no particularly obvious purpose band. Western Blot shows that the specific protein bands is in the expected location and also shows the protein expression level is not very high.Forthly, in order to verify the activity and detect the metabolites, we test the activity of CYP3A5. Midazolam and testosterone are the substrate of CYP3A4/5and can be metabolized by them. We can see that the CYP4503A5enzyme expressed in Pichia pastoris has catalytic activity and it can produce metabolite. These data is useful for the mass production of metabolites.