Expression of the secretory form of recombinant regucalcin ofBubalus bubalis in methylotropic yeast

Abstract

Regucalcin is a multifunctional protein having an important role in calcium homeostasis, L-ascorbic acid biosynthesis, anti-prolific, anti-apoptotic functions as well as detoxification of chemical warfare nerve agents. Recently, it has been localized to male reproductive tract of rat and human, and identified as an androgen-target gene. The literature suggests a possible role of regucalcin in male fertility. However, no detailed studies have been conducted on its role in male reproductive organs of domestic animals. As an initial step, we had cloned and expressed regucalcin in Pichia pastoris. The sequence analysis showed 100% homology with regucalcin of Bos tours both at nucleotide and amino acid level. The SDS-PAGE and Western blot studies of recombinant protein probed with anti-regucalcin monoclonal antibody showed a higher molecular weight (56 kDa) than the expected (35.5 kDa) that could be due to hyperglycosylation. The recombinant regucalcin and its antibodies can be used to study the detailed role of the protein in male reproduction.