Cloning, heterologous expression, and activity analysis of NADPH-cytochrome P450 reductase from the Chinese white rabbit

Abstract

Cytochrome P450 reductase (CPR) is an integral component of the P450 oxidoreductase system (P450s). It serves as the electron donor for most cytochromes in P450s, which are involved in the metabolism of foreign compounds and the synthesis of endocrine hormones and have tremendous biotechnological potential for the synthesis of pharmaceuticals and fine chemicals. However, commercially available CPR is very expensive, and heterologous expression in Escherichia coli is a more affordable way to obtain enough CPR. In the present study, a full-length cDNA encoding a CPR was isolated from the liver of the Chinese white rabbit using reverse transcription-polymerase chain reaction (RT-PCR). The cDNA contains a 2,040-bp open reading frame, which is predicted to encode an enzyme of 679 amino acids. The deduced peptide shares 99.5% amino acid similarity with CPR of Oryctolagus cuniculus, showing that the Chinese white rabbit is a close genetic relative of the European rabbit. The cloned CPR has the typical hallmarks, including an N-terminal membrane anchor and flavin adenine dinucleotide (FAD)-, flavin mononucleotide (FMN)- and nicotinamide adenine dinucleotide phosphate (NADPH)-binding domains. An N-terminally truncated protein was heterologously expressed in E. coli BL21 (DE3) cells and purified, and the specific activity of the recombinant enzyme was determined. The enzyme activity analysis indicated that electrons were passed from NADPH to Cyt C at a rate of 2.3174 μmol/(min/mg protein). The present study provides an efficient procedure for preparing large amounts of recombinant CPR, which would facilitate the synthesis of pharmaceuticals and fine chemicals with P450s.

Keywords

• Cytochrome P450 reductase
• cloning
• heterologous expression
• activity analysis
• Chinese white rabbit