Cloning and expression of koala (Phascolarctos cinereus
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- 作者：S ; ra Kong ; Suong N.T. Ngo ; Ross A. McKinnon ; Ieva Stupans
- 关键词：NADPH-cytochrome P450 reductase ; RT-PCR ; Cos-7 cells ; Sequence identity ; Protein ; mRNA level
- 刊名：Comparative Biochemistry and Physiology, Part C
- 出版年：2009
- 出版时间：July 2009
- 年：2009
- 卷：150
- 期：1
- 页码：1-9
- 全文大小：995 K
文摘
The cloning, expression and characterization of hepatic NADPH-cytochrome P450 reductase (CPR) from koala (Phascolarctos cinereus) is described. Two 2059 bp koala liver CPR cDNAs, designated CPR1 and CPR2, were cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CPR cDNAs encode proteins of 678 amino acids and share 85 % amino acid sequence identity to human CPR. Transfection of the koala CPR cDNAs into Cos-7 cells resulted in the expression of proteins, which were recognized by a goat-antihuman CPR antibody. The koala CPR1 and 2 cDNA-expressed enzymes catalysed cytochrome c reductase at the rates of 4.9 ± 0.5 and 2.6 ± 0.4 nmol/min/mg protein (mean ± SD, n = 3), respectively which were comparable to that of rat CPR cDNA-expressed enzyme. The apparent Km value for CPR activity in koala liver microsomes was 11.61 ± 6.01 µM, which is consistent with that reported for rat CPR enzyme. Northern analysis detected a CPR mRNA band of approximately 2.6 kb. Southern analysis suggested a single PCR gene across species. The present study provides primary molecular data regarding koala CPR1 and CPR2 genes in this unique marsupial species.