Cloning, prokaryotic expression and function of the Bufo bufo gargarizans 3β-hydroxysteroid dehydrogenase (3βHSD) gene

Bufadienolides, one kind of steroids, are the major active component secreted by ear-side gland of Bufo species. Preliminary studies on high-throughput transcriptome sequencing about B. bufo gargarizans showed that the expression of 3 β -Hydroxysteroid dehydrogenase (3 β HSD) in ear-side gland was nearly 20 times higher than that in liver. The enzyme 3 β HSD is an essential step in the biosynthesis of steroid such as progesterone, estrogens and androgens in steroidogenic tissues. Accordingly, 3 β HSD is probably an important enzyme involved in the biosynthesis of bufadienolides. In this study, Bbg-3βHSD cDNA was cloned from the ear-side gland of B. bufo gargarizans . Genetic engineering techniques were used to construct a recombinant prokaryotic fusion expression plasmid pCOLD- Bbg3βHSD which was introduced into E. coli BL21 for prokaryotic expression. Bbg- 3 β HSD has an open reading frame (ORF) of 1134 bp and encodes 377 amino acid residues. The speculated protein molecular weight is 42.8 kDa and its theoretical isoelectric point is 8.68. Amino acid sequence homologous analysis showed that Bbg -3 β HSD was highly homologous to the 3 β HSD protein of other species. Phylogenetic tree showed the highest similarity between Bbg -3 β HSD and 3 β HSD from Rana rugosa . The optimized expression of recombinant Bbg -3 β HSD were achieved by inducing with 0.1 mmol L −1 IPTG at 15 °C for 20 h. Enzymatic activity in vitro shows that pregnenolone and dehydroepiandroesterone could be 3 β -oxidized by Bbg -3 β HSD when NAD + was used as the coenzyme. Enzymatic properties showed that the optimum reaction temperature of recombinant Bbg- 3 β HSD was 40 °C, the optimum pH was 8.5, and the optimum coenzyme concentration was 1.5 mmol L −1 .