Research Article

Analysis of protein expression and a new prokaryotic expression system for goat (Capra hircus) spermadhesin Bdh-2 cDNA

Published: September 22, 2009
Genet. Mol. Res. 8 (3) : 1147-1157 DOI: https://doi.org/10.4238/vol8-3gmr639
Cite this Article:
J.B. Cajazeiras, L.M. Melo, E.S. Albuquerque, G. Rádis-Baptista, B.S. Cavada, V.J.F. Freitas (2009). Analysis of protein expression and a new prokaryotic expression system for goat (Capra hircus) spermadhesin Bdh-2 cDNA. Genet. Mol. Res. 8(3): 1147-1157. https://doi.org/10.4238/vol8-3gmr639
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Abstract

Low purification efficiency and incomplete characteriza­tion of male goat (buck) spermadhesins (Bdhs) prompted us to develop an effective system to produce recombinant Bdhs (rBdhs). Bdh-2 cDNA was inserted into a prokaryotic expression plasmid, pTrcHis TOPO. The pTrcHis-Bdh-2 system was constructed to produce a His6 fusion protein in Escherichia coli Top10 cells. Recombinant clones were se­lected by growth in ampicillin-enriched medium, PCR amplification and nucleotide sequencing. The inserted cDNA was completely identi­fied and recombinant protein synthesis was monitored by SDS-PAGE, followed by immunoblotting with monoclonal anti-His antibody. Ex­pression of insoluble rBdh-2 was achieved at 0.1 to 2.0 mM IPTG, after 2 to 6 h of induction. Significantly increased production of rBdh-2 (P < 0.01) occurred with 1.5 mM IPTG after 2 h of induction, and with 0.3 mM IPTG after 4 h in culture. Among the induction times investigated, a period of 6 h gave the lowest levels of rBdh-2 production; with a 6-h incubation, there were no significant differences in rBdh-2 production for the various concentrations of IPTG tested (P > 0.05). The apparent molecular weight of rBdh-2 was 15.85 ± 0.09 kDa, calculated by im­age analysis of membranes. This is similar to the theoretical molecu­lar weight of 15.5 kDa predicted from the nucleotide sequence. Prior to this study, expression of recombinant goat spermadhesin had never been reported. Thus, an effective prokaryotic rBdh-2 expression system was developed in order to provide an adequate tool for studying bio­functions of goat spermadhesins.

Low purification efficiency and incomplete characteriza­tion of male goat (buck) spermadhesins (Bdhs) prompted us to develop an effective system to produce recombinant Bdhs (rBdhs). Bdh-2 cDNA was inserted into a prokaryotic expression plasmid, pTrcHis TOPO. The pTrcHis-Bdh-2 system was constructed to produce a His6 fusion protein in Escherichia coli Top10 cells. Recombinant clones were se­lected by growth in ampicillin-enriched medium, PCR amplification and nucleotide sequencing. The inserted cDNA was completely identi­fied and recombinant protein synthesis was monitored by SDS-PAGE, followed by immunoblotting with monoclonal anti-His antibody. Ex­pression of insoluble rBdh-2 was achieved at 0.1 to 2.0 mM IPTG, after 2 to 6 h of induction. Significantly increased production of rBdh-2 (P 0.05). The apparent molecular weight of rBdh-2 was 15.85 ± 0.09 kDa, calculated by im­age analysis of membranes. This is similar to the theoretical molecu­lar weight of 15.5 kDa predicted from the nucleotide sequence. Prior to this study, expression of recombinant goat spermadhesin had never been reported. Thus, an effective prokaryotic rBdh-2 expression system was developed in order to provide an adequate tool for studying bio­functions of goat spermadhesins.