Cloning And Expression Of CYP2D6 Gene And Its Application In Drug Selection

The molecular mechanism of human CYP2D6 polymorphism and the influence on drug metabolism are both reserch hotspot in last years. More than 70 of CYP2D6 SNPs were indentified. Although lots of CYP2D6 data have been reported, Most of those studied CYP450 from a allele to another . Due to difference of enzyme preparation, probe substrates and analysis, the data and conclusion from CYP2D6 phenotype was very antilogy. It was very difficult to explain the final result.Human CYP2D6 is responsible for the metabolism of 30% drugs. In order to study the effects of single nucleotide polymorphism on CYP2D6, 8 non-synonymous SNPs (V7M, V11M, P34S, G42R, G212E, L213S, P325L, L421P) have been chosen.1. Cloning CYP2D6 geneAccording to CYP2D6 wildtype cDNA sequence in gene database, the primers of CYP2D6 wildtype had been designed. CYP2D6 wild type cDNA had been obtained by RT-PCR, then cDNA fragment and pYES2/CT vector were chipped by restricted enzyme .Both of them were ligased together. Consequently the recombined plasmid was built successfully. 8 non-synomymous SNPs were obtained by specific-site mutagenesis PCR, then cloned into pYES2/CT vector in the same method. The 8 non-synonymous recombition plasmids could expressed in yeast (Saccharomyces cerevisiae).2. Expressing CYP2D6 recombinated plasmidsCYP2D6 wild type and 8 mutants separately transformed into yeast which is auxotroph yeast INVScl, then expressed protein by using galatcose induction in auxotroph medium SG-U. Harvested yeast cells , and ultrasonicated them, finally yeast microsomes which had CYP2D6 recombinated enzyme were obtained, were confirmed by Western-Blotting.3. A large scale induction of CYP2D6 and analysis the expressed proteinFollowing the same culture condition, a large scale CYP2D6 proteins were expressed, and were crashed yeast cells by ultrasorniation, then obtained yeast microsome which contains a lot of CYP2D6 enzyme proteins. Detected absorption at 450nm weavelenght which is indirectly reflecting the content of CYP2D6 protein in yeast microsome by using the CO—differet spectrum detecting method. 4. Enzyme activity confirmationIn NADPH reganation system added microsomes containing CYP2D6 protein, and also added a range of different diluted substrate AMMC solutions. Detected CYP2D6 enzyme activity, calculate all kinetic contents including CYP2D6 wild type and 8 non-synonymous SNPs. Compared with CYP2D6 wild type , V7M,V11M,P325L mutants revealed normal activity and P34 mutant showed low activity, but the mutants of G42R,P34S,G212E,L213S,P325L,L421P lost activity. By comparing the structrue between CYP2D6 wild type and mutants, the amino acid residue changing had some effects on the structure of the protein.5. Inhibition of some drugs to CYP2D6 enzyme activityAs an important subfamily of CYP450 enzyme, CYP2D6 can word on more than 100 significant drug metabolism. CYP2D6 can also inhibite competitively with some drugs. All of these caused different individual drug dose, and drug interactions. Study different drugs inhibition to CYP2D6 enzyme activity, contenting antipsychotic drug, anti-arrhythmic drug, antidepressant drug, hypotenso drug, analgesic drug, antifungal drug, antacid drug, antidiabetic drug, anti-arteriosclerosis drug, anticoagulant drug, anti-angina drug using a in-vitro model. Preliminary summarize drug and CYP2D6 enzyme relationship about inhibition, and successfully construct a dectecting method of drug-CYP2D6 enzyme inhibiton.In the end, this research successfully expressed human CYP2D6 recombinate enzyme (wild type and 8 non-synonymous SNPs) which had catalytic activity in Saccharomyces cerevisiae system, that can use as a powerful tool for in-vitro drug metabolism research , and preliminary construct a dectecting method of drug-CYP2D6 enzyme inhibiton.