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

Abstract

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 His 6 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.

Producción Y Purificación De Osteopontina Bovina Recombinante Mediante Escherichia coli Como Fábrica Celular

“…En este sentido, lograr su producción de forma recombinante, nos facilita conocer sus características estructurales y funcionales, además contribuye a dilucidar su participación en los procesos reproductivos. Al respecto, la fábrica celular seleccionada fue E.coli, debido a su versatilidad, múltiples opciones genéticas y fácil cultivo, así como costos de los medios (Cajazeiras et al, 2009).…”

Recombinant expression of porcine spermadhesin AWN and its phospholipid interaction: Indication for a novel lipid binding property

“…Here, we generated a transfected E. coli strain and produced for the first time recombinantly expressed porcine spermadhesin AWN. This expands the range of recombinant spermadhesins already including Bodhesin‐2 from goat (Cajazeiras et al., ) porcine AQN‐1 and bovine spermadhesin‐1 (referred to as aSFP, Ekhlasi‐Hundrieser, Calvete, et al., ). A three ‐ step protocol was successfully developed to purify this His‐tagged AWN in sufficient purity for functional studies.…”

Recombinant Feline Oviductin – A Powerful Tool for Functional IVF Studies in the Domestic Cat

“…) and goat (Cajazeiras et al. ). Studies with oviductin produced in E. coli without post‐translational modifications might elucidate the influence of the oviductin carbohydrate side chains on reproductive processes.…”

“…If the purified protein is available in sufficient quantities, we will investigate whether the recombinant protein is able to improve IVF and/or embryo culture in the domestic cat. Functional studies with recombinant proteins, relevant for reproductive research, have already been performed for example in human (Garde et al 2007) and goat (Cajazeiras et al 2009). Studies with oviductin produced in E. coli without post-translational modifications might elucidate the influence of the oviductin carbohydrate side chains on reproductive processes.…”