Expression of recombinant buck (Capra hircus) spermadhesinin a prokaryotic system

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-4 cDNA was inserted in a prokaryotic expression plasmid pTrcHis TOPO 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 recombinant protein synthesis was monitored by SDS-PAGE followed by immunoblotting using a monoclonal anti-His antibody. The expression of the rBdh-4 was achieved at 0.1 to 2.0 mM IPTG after 2 to 6 h of induction. A greater production of rBdh-4 (P < 0.001) was obtained with 0.1 mM IPTG after 2 h of induction. The apparent molecular weight of rBdh-4 was 15.85 ± 0.09 kDa. This result agrees with the theoretical molecular weight of 16.5 kDa predicted from the nucleotide sequence. In conclusion, an effective rBdh-4 expression system was established in order to provide a good tool for studying the biofunctions of buck spermadhesins.(Expressão da espermadesina recombinante de bode (Capra hircus) em sistema procariótico). A baixa eficiência de purificação e a incompleta caracterização das espermadesinas de bode (Bdhs) nos levou a estabelecer um sistema efetivo para produzir as Bdhs recombinantes (rBdhs). O cDNA da Bdh-4 foi inserido no plasmídeo de expressão procariótico pTrcHis TOPO para produzir uma proteína de fusão His6 em células de E.coli Top10. Os clones recombinantes foram crescidos em meio contendo ampicilina, amplificação por PCR e sequenciamento de nucleotídeos. A síntese da proteína recombinante foi monitorada por SDS-PAGE seguida por imunobloting usando anticorpo monoclonal anti-His. A expressão da rBdh-4 foi conseguida de 0,1 a 2,0 mM de IPTG e depois de 2 a 6 H de indução. A maior produção da rBdh-4 (

Expression of recombinant buck (Capra hircus) spermadhesinin a prokaryotic system

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-4 cDNA was inserted in a prokaryotic expression plasmid pTrcHis TOPO 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 recombinant protein synthesis was monitored by SDS-PAGE followed by immunoblotting using a monoclonal anti-His antibody. The expression of the rBdh-4 was achieved at 0.1 to 2.0 mM IPTG after 2 to 6 h of induction. A greater production of rBdh-4 (P < 0.001) was obtained with 0.1 mM IPTG after 2 h of induction. The apparent molecular weight of rBdh-4 was 15.85 ± 0.09 kDa. This result agrees with the theoretical molecular weight of 16.5 kDa predicted from the nucleotide sequence. In conclusion, an effective rBdh-4 expression system was established in order to provide a good tool for studying the biofunctions of buck spermadhesins.(Expressão da espermadesina recombinante de bode (Capra hircus) em sistema procariótico). A baixa eficiência de purificação e a incompleta caracterização das espermadesinas de bode (Bdhs) nos levou a estabelecer um sistema efetivo para produzir as Bdhs recombinantes (rBdhs). O cDNA da Bdh-4 foi inserido no plasmídeo de expressão procariótico pTrcHis TOPO para produzir uma proteína de fusão His6 em células de E.coli Top10. Os clones recombinantes foram crescidos em meio contendo ampicilina, amplificação por PCR e sequenciamento de nucleotídeos. A síntese da proteína recombinante foi monitorada por SDS-PAGE seguida por imunobloting usando anticorpo monoclonal anti-His. A expressão da rBdh-4 foi conseguida de 0,1 a 2,0 mM de IPTG e depois de 2 a 6 H de indução. A maior produção da rBdh-4 (

Effect of Lectins from Diocleinae Subtribe against Oral Streptococci

Surface colonization is an essential step in biofilm development. The ability of oral pathogens to adhere to tooth surfaces is directly linked with the presence of specific molecules at the bacterial surface that can interact with enamel acquired pellicle ligands. In light of this, the aim of this study was to verify inhibitory and antibiofilm action of lectins from the Diocleinaesubtribe against Streptococcus mutans and Streptococcus oralis. The inhibitory action against planctonic cells was assessed using lectins from Canavaliaensi formis (ConA), Canavalia brasiliensis (ConBr), Canavalia maritima (ConM), Canavalia gladiata (CGL) and Canavalia boliviana (ConBol). ConBol, ConBr and ConM showed inhibitory activity on S. mutans growth. All lectins, except ConA, stimulated significantly the growth of S. oralis. To evaluate the effect on biofilm formation, clarified saliva was added to 96-well, flat-bottomed polystyrene plates, followed by the addition of solutions containing 100 or 200 µg/mL of the selected lectins. ConBol, ConM and ConA inhibited the S. mutans biofilms. No effects were found on S. oralis biofilms. Structure/function analysis were carried out using bioinformatics tools. The aperture and deepness of the CRD (Carbohydrate Recognition Domain) permit us to distinguish the two groups of Canavalia lectins in accordance to their actions against S. mutans and S. oralis. The results found provide a basis for encouraging the use of plant lectins as biotechnological tools in ecological control and prevention of caries disease