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

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 3β-Hydroxysteroid dehydrogenase (3βHSD) in ear-side gland was nearly 20 times higher than that in liver. The enzyme 3βHSD is an essential step in the biosynthesis of steroid such as progesterone, estrogens and androgens in steroidogenic tissues. Accordingly, 3βHSD 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-3βHSD 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-3βHSD was highly homologous to the 3βHSD protein of other species. Phylogenetic tree showed the highest similarity between Bbg-3βHSD and 3βHSD from Rana rugosa. The optimized expression of recombinant Bbg-3βHSD 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 3β-oxidized by Bbg-3βHSD when NAD⁺ was used as the coenzyme. Enzymatic properties showed that the optimum reaction temperature of recombinant Bbg-3βHSD was 40 °C, the optimum pH was 8.5, and the optimum coenzyme concentration was 1.5 mmol L^-1.