Espermadesinas caprinas caprinas (bodesinas): produção em sistema bacteriano. / Espermadesinas goat (bodesinas) production in bacterial system.

AUTOR(ES)
FONTE

IBICT - Instituto Brasileiro de Informação em Ciência e Tecnologia

DATA DE PUBLICAÇÃO

28/08/2009

RESUMO

The low purification efficiency and the incomplete characterization of buck spermadhesins (Bdhs) prompted us to establish an effective system to produce recombinant Bdhs (rBdhs). The Bdh-2 and Bdh-4 cDNA was inserted in a prokaryotic expression plasmid pTrcHis TOPO. The pTrcHis-Bdh system was constructed to produce a His6 fusion protein in E. coli Top10 cells. The recombinant clones were selected by growth in ampicillin-containing medium, PCR amplifications and nucleotide sequencing. The inserted cDNA was completely identified and the recombinant protein synthesis was monitored by SDS-PAGE followed by immunoblotting using monoclonal anti-His antibody. The expression of both insoluble rBdhs was achieved at 0.1 to 2.0 mM IPTG and after 2 to 6 h of induction. A greater production of both rBdhs occurred with 1.5 mM IPTG after 2 h of induction, and with 0.3 mM IPTG after 4 h of culture. Among the induction times investigated, 6 h showed the lowest levels of rBdh-2 and Bdh-4 production, where no difference was seen between the various concentrations of IPTG tested (P >0.01). The apparent molecular weight of rBdh-2 was 15.85 0.09 kDa and rBdh-4 was 18.17 0.03 kDa calculated by image analysis of membranes. This result agrees with the theoretical molecular weight of 15.5 kDa for Bdh-2 and 16,5 KDa for Bdh-4 predicted from the nucleotide sequence. Prior to this study, expression of recombinant buck spermadhesin had never been reported. Thus, an effective prokaryotic rBdh expression system was established in order to provide a good tool for studying the biofunctions of buck spermadhesins.

ASSUNTO(S)

caprino espermadesina proteína recombinante reproducao animal goat spermadhesin recombinant protein

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