Chicken Cytochrome P4501A5、1A4Heterologous Expression And Activity Assay In Vitro

More and more attention of cytochrome P450have been paied in drug bioavailability, drug interactions, and roles in the formation of carcinogenic metabolites. Research in this area has mainly focused on human and some laboratory animal. The metabolism of drugs and other endogenous compounds by cytochrome P450of chicken hasn’t been explored in depth. Liver is the organ with most abundant P450regarding both types and level. Traditional in vitro drug metabolism studies use liver microsomes or liver cells, but various forms of P450are present in an animal liver, and their structures possess high similarity. Furthermore, separation and purification of a single P450protein are both complicated. With the advance of molecular bio-technology, the recombinant expression of P450genes in toxicology, pharmacology and other research areas have been becomed essential,which enabled reseachers to gain understandings of the functions of P450in toxicology and pharmacology.To survey the in vitro activities of chicken cytochrome P4501A5and1A4(CYP1A5)(CYP1A4), genetically engineered Escherichia coli was used in the present study for recombinant expression of target proteins. Chicken cDNA was used as template to amplify CYP1A5and CYP1A4genes cDNA which are then inserted into pCW vector (EcoR Ⅰ—SalⅠ). Expression of recombinant proteins was then induced by IPTG with engineered E. coli transformed by recombinant plasmids. As proved by CO-difference spectra, His-CYP1A5and His-CYP1A4had the typical P450absorption peak. The recombinant His-CYP1A5reconstituted with cytochrome P450reductase (CPR) showed7-ethoxyresorufin O-deethylase activity. The results suggested that in the present study recombinant chicken cytochrome P4501A5(CYP1A5) and1A4(CYP1A4) proteins with catalytic activity have been successfully expressed.