| The cytochrome P450(CYP) enzymes constitute a superfamily of heme-containing mono-oxygenases that catalyse metabolism with endogenous including steroids, bile acids, prostaglandins, arachidonic acid, biological source, and a wide variety of xenobiotics including drugs, plant derived or fungal-derived secondary metabolites consumed with food, and a large number of environmental pollutants, industrial compounds, herbicides, and pesticides. The human CYP forms that metabolize xenobiotics belong to the families CYP1, CYP2and CYP3. Individual CYP enzymes in these families have broad and overlapping substrate specificities, and are responsible for the metabolism of approximately70-80%of all currently used drugs.The human cytochrome P450(CYP) enzymes play a major role in the metabolism of endobiotics and numerous xenobiotics including drugs. Therefore it is the standard procedure to test new drug candidates for interactions with CYP enzymes during the preclinical development phase. Modulation of CYP activity via inhibition or induction by drugs and other xenobiotics often is the source of drug interactions. Drug interactions can evoke severe adverse effects, they have resulted in early termination of drug development, refusal to obtain approval, severe prescribing restrictions, and even withdrawal of drugs from the market. The most common mechanism of drug interactions is inhibition of these CYP enzymes.In early drug development, experiments are routinely carried out to determine which CYP enzymes catalyse the metabolism of lead compounds. In addition, the potential of lead compounds to inhibit CYPs can be evaluated with in vitro methods. Often, but not always, a compound that inhibits a specific CYP form is also a substrate for that same form.CYP3A4is one of the very important cytochrome P450in human body, accounting for about50%of total CYPs. CYP3A4can be induced or inhibited by many drugs, with gene polymorphism, which is one of the most important factors in causing individual differnernce for drug effects, also be one of the causes of drug interactions. CYP3A4has a pivotal role in xenobiotic metabolism, and it has been estimated to be involved in the metabolism of up to50%of all drugs in clinical use. The active site of CYP3A4is very large and flexible allowing the binding and subsequent metabolism of structurally very diverse compounds. The substrate binding is principally based on hydrophobicity with some steric interactions.E.coli is the most frequently used host for the recombinant expression of human CYPs. Besides the easy genetic manipulation and cultivation, E.coli is a suitable host for the expression of CYP since it has no own CYP background. Also the protein yield in E.coli is very high and thus the expression level of recombinant CYPs can be50-100times higher in E. coli compared to that in S. cerevisiae. Generally, the expression levels of human CYPs expressed in E.coli that can be found in literature vary in a notable range. This can be ascribed to the usage of different expression hosts or genetic strategies. The expression levels of human CYPs in E.coli in particular depend on the culture conditions during cultivation of these E. coli strains.Mammalian P450protein was first expressed in1991, via the modification of the N-terminal amino acid sequences in E. coli cells. Since that time, a variety of strategies have been established for the functional expression of recombinant P450s in E. coli, including N-terminal modification, the use of molecular chaperones, and culturing at lower temperatures. In all cases, human P450expressed in E. coli cells has been shown to efficiently catalyze the oxidation of representative substrates at efficient rates. These recombinant P450s are applicable to studies which estimate the kinetic parameters of drug oxidation, and have also been used to determine the metabolic pathways of drugs and carcinogens exploited by human P450s. It is now possible to utilize a variety of recombinant human enzymes for various purposes, and in the development of a new pharmaceutical drug candidate, it is almost routine to determine which of the P450s contribute to their metabolism. This approach has been utilized in the making of rational in vitro predictions regarding bioavailability, toxicity, and drug-drug interactions.Objective Comparison the effect of two stored method for tissue total RNA by saline or RNAlater solution, in order to get a good prervation method of liver tissue.Investigate the effect of alpha-ALA (0.5mM and1mM), IPTG (0.5mM and1mM), concentration of kanamycin (50μg/mL and100μg/mL) and bacteria inoculation dendity on CYP3A4membrane protein expression levels, conditions for the optimization of the reorganization of CYP3A4in E.coli.To construct the recombinant the human CYP3A4in E. coli, in order to study drugs interaction in vitro, which can also be used for determine the metabolic characteristics of drugs and drugs interaction when new drugs development.To obtain CYP3A4variation by site-directed mutagenesis on the basis of recombinant wild-type CYP3A4, in order to study the differences between wild-type and variation for drug metabolism in vitro, that can be used to guide personalized medicine in clinical.Methods(1)Human liver total RNA extraction:Liver tissues were collected with saline or RNAlater solution processing stored at-20℃(tissue of preservation by saline at-80℃). The totle RNA from liver tissue was extracted by TRIZOL (?) Reagent. The concentration and purity of total RNA were determined, to compare two save method for total RNA. The integrity of total RNA was verified.(2) Cloning, modification and identification of CYP3A4gene:The target gene of CYP3A4was obtained by RT-PCR using total RNA as template. CYP3A4cDNA was purified by gel extraction kit, then was connected to pMD (?)20-T vector, following transformed into E. coli by heat shock. After Blue-white screening and colonu PCR, the target gene was modified for N-terminal(MALLLAVF)and C-terminal His (5) plus, then was connected to pMD (?)20-T vector, and transformed into E. coli, which was screened by blue-white colony and PCR validation, before verified by sequencing. The correct sequenced of recombinant T vector (containing the CYP3A4gene) was digested by NcoI and Xhol and then connected to pET-28a(+) with the same digested, and transformed into E. coli, which was screened by blue-white colony and PCR validation, before verified by sequencing.(3) According to the Takara MutanBEST Kit (D401) site-directed mutagenesis, principles were designed for CYP3A4mutation subtype of CYP3A4*3, CYP3A4*15, CYP3A4*17, CYP3A4*18and CYP3A4*19. Site-directed mutagenesis was carried out by Takara MutanBEST Kit and two overlapping PCR method respectively. The mutated PCR products were purified by gel extraction kit, then connected into expression vector and transformed into E. coli. The mutagenesis was detected by sequencing.(4) The correct sequenced of recombinant expression vector pET-3A4was transformed into expression strain E.coli BL21(DE3). Four factor and two levels of orthogonal experiment designed by SPSS13.0to optimized four factors of a-ALA (0.5mM and1mM), IPTG (0.5mM and1mM), kanamycin (50μg/mL and100μg/mL) concentration and bacteria inoculation density (inoculation1%and inoculated with2%) for portent expression. The results were analyzed using SPSS13.0to select a good combination of large-scale induced expression. After induction, bacteria was collected by centrifuged, and then broken by Ultrasonication treatment, centrifuged, the supernatant was carefully transferred to the EP tube, suspended by PB solution, stored at-80℃. The extraction of membrane proteins was analyzed by BCA protein concentration quantitative method. Extraction of membrane proteins was verified by Western blot whether the desired target protein.Result(1)The concentration of extracted total RNA treated by RNAlater is more than300μg/gFW purity in OD260/OD280of1.96±0.015(pure RNA OD260/OD280value of2.0), that is significantly higher than treated by saline. The integrity of total RNA treated by RNAlater is better than saline.(2) Validation for CYP3A4gen by sequencing is correct. And the modification of the N-terminal and C-terminal is verified by sequencing. Recombinant expression vector pET-3A4is also verified by sequencing.(3) CYP3A4*3,*15*17*18*19subtypes by Site-directed mutagenesis are verified by PCR, but there only show one subtypes (CYP3A4*19) with correct mutations by sequencing(4) Membrane protein concentration is around65μg/mL. The level of a-ALA, antibiotics (kanamycin), IPTG, inoculation density on the level of expression of membrane proteins was no statistically significant. Expression of membrane proteins was verified by Western blot for recombinant CYP3A4protein.ConclusionSuccessfully constructed the expression vector of pET-3A4, and obtained a subtype of CYP3A4CYP3A4*19clone. After the optimization of induction conditions, we obtain CYP3A4membrane protein, and verify the specificity of membrane proteins by Western blot for the reorganization of CYP3A4, that is the basis of identifing recombinant CYP3A4enzyme activity and application for drug interactions.The subject to overcome expression of recombinant cytochrome enzyme must use generic expression vector pCW. Recombinant cytochrome enzymes CYP3A4was successfully expressed by using PET-28a(+). |
PDF Full Text Request