cDNA-directed expression of a functional zebrafish CYP1A in yeast.
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 degrees 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 Km (Km(app)) and Vmax values at 30 degrees C of 0.31 +/- 0.04 microM and 0.70 +/- 0.10 nmol/min/nmol CYP, respectively. The recombinant protein also metabolized benzo(a)pyrene with a Km(app) and Vmax values of 5.34 +/- 0.58 microM 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.
Chung
Woon-Gye
WG
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR, USA.
Sen
Alaattin
A
Wang-Buhler
Jun-Lan
JL
Yang
Yea-Huey
YH
Lopez
Nathan
N
Merrill
Gary F
GF
Miranda
Cristobal L
CL
Hu
Chin-Hwa
CH
Buhler
Donald R
DR
D000818:Animals; D001564:Benzo(a)pyrene / Q000378:metabolism; D003001:Cloning, Molecular; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D008862:Microsomes, Liver / Q000201:enzymology; D012313:RNA / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D015027:Zebrafish / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D001564:Benzo(a)pyrene; D012313:RNA; D019363:Cytochrome P-450 CYP1A1