Expressão, purificação e caracterização estrutural da bdh-2 recombinante, uma espermadesina presente no plasma seminal de bode (Capra hircus)

Abstract

The Bdh-2 bodhesin cDNA present in seminal plasma of goat was subcloned in the expression plasmid pTrcHis TOPO used to transform E. coli Top10 One shot cells. The recombinant clones were selected by growth in 50 μg/mL ampicillin-containing LB-Broth medium and PCR amplifications. The recombinant protein synthesis was monitored by SDS-PAGE followed by immunoblotting using monoclonal anti-His antibody. The production of the rBdh-2 through low temperature was not satisfactory. A greater production of the rBdh-2 occurred with IPTG 1.5 mM after 2 h of induction. The method utilized to the purification of the rBdh-2 was realized in affinity chromatography on a His-Trap column following ion-exchange chromatography on a DEAE-Sephacel column. The secondary structure of the rBdh-2 was evaluated through spectral profile circular dichroism (CD) and confirmed the prevalence of secondary structures like β-sheets, the presence of a low content of unfolded structures and helices-. The rBdh-2 structure remained stable up to 35 °C. However, significant changes were observed from 40 °C related to a distortion of the spectrum of CD.