cDNA-directed expression of a functional zebrafish CYP1A in yeast

Abstract

A cytochrome P450 1A (CYP1A) cDNA was isolated from an adult zebrafish (Danio rerio) library. The 2580-bp clone (GenBank Accession No. AF210727) contained a 62-bp 5'-unstranslated region (UTR), 1557-bp coding region and 962-bp 3'-UTR. The deduced 519-residue protein (calculated molecular weight 58,556, pI = 7.58) shared 74% identity with rainbow trout CYP1A and 57 and 54% identities with mouse and human CYP1A1s, respectively. The zebrafish CYP1A protein coding region was cloned into the pDONR201 entry vector and then transferred to a yeast expression vector pYES-DEST52. Expression of zebrafish CYP1A in Saccharomyces cerevisiae transformants was induced by galactose to a maximum level of 493 pmol CYP1A per mg microsomal protein or about 8 nmol/l of culture. Recombinant CYP1A protein expressed in yeast was mainly in the denatured P420 form under normal microsomal preparation conditions but when the oxygen concentration was reduced in the buffer by degassing and the yeast cells were maintained at less than 10°C, the integrity of the CYP1A was preserved and it exhibited a characteristic reduced CO-difference spectrum maximum at 448 nm. The recombinant zebrafish CYP1A demonstrated 7-ethoxyresorufin O-deethylase (EROD) activity with an apparent K m (Kmapp) and V max values at 30°C of 0.31 ± 0.04 µM and 0.70 ± 0.10 nmol/min/nmol CYP, respectively. The recombinant protein also metabolized benzo(a)pyrene with a Kmapp and V max values of 5.34 ± 0.58 µM and 1.16 ± 0.13 nmol/min/nmol CYP, respectively. These results show the recombinant expression of a functional zebrafish CYP in yeast and validated yeast as a host for heterologous expression of zebrafish CYP1A and potentially for other zebrafish CYPs. © 2004 Elsevier B.V. All rights reserved.