cDNA cloning and functional expression in yeast Saccharomyces cerevisiae of β‐naphthoflavone‐induced rabbit liver P‐450 LM4 and LM6

Abstract

A cDNA library was constructed from liver mRNA of a β‐naphthoflavone‐induced rabbit. Two clones pLM4‐1 and pLM6‐1 containaing 2.2‐kbp inserts that hybridized at low stringincy with a mouse P1 P‐450 probe were selected. The clone pLM4‐1 was fully sequenced and found to contain a full‐length cDNA coding for cytochrome P‐450 LM4. Partial sequence and restriction mapping made it possible to identify pLM6‐1 as coding for the major part of cytochrome P‐450 LM6. Cloned LM4‐1 cDNA was reformed by deletion of the 5′ and 3′ non‐coding regions before insertion into yeast expression vectors PYe DP1/10. A similar operation was performed on pLM6–1 cDNA after replacement of the missing N‐terminus‐coding sequences by homologous sequences form the pLM4–1 clone resulting in a chimeric cytochrome P‐450 coding sequence. Expression of cloned rabbit cytochrome P‐450 into transformed yeast was optimized by studying the effect of the nature of the DNA sequence just preceding the initiation codon on the level of cytochrome P‐450 production. Yeast synthesized cytochromes P‐450 were characterized by immunoblotting, spectra and catalytic activity determinations. Cloned cytochrome P‐450 LM4 was found by all criteria to be identical to the authentic rabbit one. The chimeric cytochrome P‐450 that contains the 143 N‐terminal amino acids of cytochrome P‐450 LM4 and the remaining 375 amino acids of cytochrome P‐450 LM6 was found to exhibit most of the authentic cytochrome P‐450 LM6 catalytic properties. Enzymatic and evolutionary implications of these results are discussed.