PMID- 15522429
OWN - NLM
STAT- MEDLINE
DCOM- 20041223
LR  - 20131121
IS  - 0166-445X (Print)
IS  - 0166-445X (Linking)
VI  - 70
IP  - 2
DP  - 2004 Nov 18
TI  - cDNA-directed expression of a functional zebrafish CYP1A in yeast.
PG  - 111-21
AB  - 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.
FAU - Chung, Woon-Gye
AU  - Chung WG
AD  - Department of Environmental and Molecular Toxicology, Oregon State University, 
      Corvallis, OR, USA.
FAU - Sen, Alaattin
AU  - Sen A
FAU - Wang-Buhler, Jun-Lan
AU  - Wang-Buhler JL
FAU - Yang, Yea-Huey
AU  - Yang YH
FAU - Lopez, Nathan
AU  - Lopez N
FAU - Merrill, Gary F
AU  - Merrill GF
FAU - Miranda, Cristobal L
AU  - Miranda CL
FAU - Hu, Chin-Hwa
AU  - Hu CH
FAU - Buhler, Donald R
AU  - Buhler DR
LA  - eng
GR  - ES 00210/ES/NIEHS NIH HHS/United States
GR  - ES 11587/ES/NIEHS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - Netherlands
TA  - Aquat Toxicol
JT  - Aquatic toxicology (Amsterdam, Netherlands)
JID - 8500246
RN  - 0 (DNA, Complementary)
RN  - 0 (Recombinant Proteins)
RN  - 3417WMA06D (Benzo(a)pyrene)
RN  - 63231-63-0 (RNA)
RN  - EC 1.14.14.1 (Cytochrome P-450 CYP1A1)
SB  - IM
MH  - Animals
MH  - Benzo(a)pyrene/metabolism
MH  - Cloning, Molecular
MH  - Cytochrome P-450 CYP1A1/biosynthesis/*genetics/metabolism
MH  - DNA, Complementary/genetics
MH  - Gene Library
MH  - Microsomes, Liver/enzymology
MH  - RNA/chemistry/genetics
MH  - Recombinant Proteins
MH  - Reverse Transcriptase Polymerase Chain Reaction/veterinary
MH  - Saccharomyces cerevisiae/enzymology/*genetics
MH  - Zebrafish/*genetics/metabolism
EDAT- 2004/11/04 09:00
MHDA- 2004/12/24 09:00
CRDT- 2004/11/04 09:00
PHST- 2004/06/10 00:00 [received]
PHST- 2004/07/20 00:00 [revised]
PHST- 2004/07/29 00:00 [accepted]
PHST- 2004/11/04 09:00 [pubmed]
PHST- 2004/12/24 09:00 [medline]
PHST- 2004/11/04 09:00 [entrez]
AID - S0166-445X(04)00239-5 [pii]
AID - 10.1016/j.aquatox.2004.07.007 [doi]
PST - ppublish
SO  - Aquat Toxicol. 2004 Nov 18;70(2):111-21. doi: 10.1016/j.aquatox.2004.07.007.