A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2 % glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.
The expressed sequence tag (EST) effort in Toxoplasma gondii has generated a substantial amount of gene information. To exploit this valuable resource, we chose to study tgd057, a novel gene identified by a large number of ESTs that otherwise show no significant match to known sequences in the database. Northern analysis showed that tgd057 is transcribed in this tachyzoite. The complete cDNA sequence of tgd057 is 1169 bp in length. Sequence analysis revealed that tgd057 possibly adopts two polyadenylation sites, utilizes the fourth in-frame ATG for translation initiation, and codes for a secretory protein. The longest open reading frame for the tgd057 gene was cloned and expressed as a recombinant protein (rd57) in Escherichia coli. Western analysis revealed that serum against rd57 recognized a molecule of ~21 kDa in the tachyzoite protein extract. This suggests that the tgd057 gene is expressed in vivo in the parasite.
The scFv antibody towards the Burkholderia pseudomallei exotoxin was previously constructed by phage display and exhibited good specificity towards the exotoxin. We report here the optimization of the scFv expression in an E. coli expression system. Four different E. coli strains (ER2537, TG1, HB2151, and XL1-Blue) were examined for optimal expression of the scFv protein. Two types of carbon source (i.e. 0.2% glucose and 0.2% glycerol) were also tested for their ability to induce the scFv expression. Cells that carried the scFv construct were grown at 30 degrees C and induced with 0.05 mM IPTG. The expression was then monitored by SDS-PAGE, Western blotting, and indirect ELISA. The Western blot profile showed different levels of the scFv expression among the host strains; XL1-Blue exhibited the highest level of the scFv protein expression. Glycerol at a concentration of 0.2% (v/v) significantly increased the scFv protein expression level when compared to 0.2% (w/v) glucose. Further optimization demonstrated that the scFv protein expression in XL1-Blue was the most optimal with a glycerol concentration as low as 0.05%. However, by indirect ELISA, only the scFv protein that was expressed in 0.2% (v/v) glycerol exhibited high specificity towards the Burkholderia pseudomallei exotoxin.
In the fight against cancer, novel chemotherapeutic agents are constantly being sought to complement existing drugs. Various studies have presented evidence that the apoptosis that is induced by these anticancer agents is implicated in tumor regression, and Bcl-2 family genes play a part in apoptosis following treatment with various stimuli. Here, we present data that a styrylpyrone derivative (SPD) that is extracted from the plant Goniothalamus sp. showed cytotoxic effects on the human breast cancer cell line MCF-7. SPD significantly increased apoptosis in MCF-7 cells, as visualized by phase contrast microscopy and evaluated by the Tdt-mediated dUTP nick end-labeling assay and nuclear morphology. Western blotting and immunostaining revealed up-regulation of the proapoptotic Bax protein expression. SPD, however, did not affect the expression of the anti-apoptotic protein, Bcl-2. These results, therefore, suggest SPD as a potent cytotoxic agent on MCF-7 cells by inducing apoptosis through the modulation of Bax levels.