Browsing by Author "Wu, Rui"

Saccharomyces cerevisiae var. boulardii is a probiotic strain of the baker’s yeast Saccharomyces cerevisiae. It has a long history of use in treating and preventing several kinds of diarrhea in human. Recently, scientists have drawn their attentions to use this probiotic yeast as a living drug delivery vehicle to the gastric intestinal tract (GIT). Several distinctive features of S. boulardii such as an optimal growth temperature at 37 ºC and better acidic tolerance enable active recombinant protein expression in harsh conditions. Human intrinsic factor (IF) is a glycoprotein secreted by gastric parietal cells and belongs to vitamin B12 (Cobalamin, Cbl) transport family. The gastric IF gene (GIF) encodes this protein. It is an essential protein for the proper absorption of Cbl in the terminal ileum, lacking of which results in vitamin B12 deficiency, especially common among the elders. This study aims to express active human IF from probiotic S. boulardii that can be combined with vitamin B12 forming B12/IF complex and absorbed by intestinal cells. To achieve these goals, a plasmid pSF-TEF1-TPI1-Blast-GIF was designed for S. boulardii transformation. Verification of recombinant IF was accomplished by SDS-PAGE, Western blotting and peptide sequence mapping by mass spectrometry (MS). Experimental results indicated that the recombinant IF was not able to be synthesized in useful amount from S. boulardii using plasmid pSF-TEF1-TPI1-Blast-GIF. Transformation of the plasmid carrying GIF sequence brought significant phenotypic and metabolic modification to the host cells. PCR technique was found unsuccessful to verify the presence of correct plasmid, leaving uncertainty whether the correct plasmid was present in the transformants or not. Interestingly, an immunreoactive band at expected size (45 kDa) could be detected with rat polyclonal antibody in Western blotting. This band could be only detected from the supernatant when cells were grown with Cbl supplement, but shown to be exo-1,3-β-glucanase in MS protein sequencing. However, it is still possible that trace amount of recombinant IFs are secreted but cannot be detected due to MS sequencing limitations. Even though there was a contradictory result in Western blotting compared to that of MS sequencing, the excessive secretion of the exo-1,3-β-glucanase which may corrode yeast cell walls, may explain the different phenotype of transformants in comparison to host cells. This study indicated that not all genetic manipulation tools designed for S. cerevisiae are without problems for S. boulardii. More studies need to be carried out for successful heterologous IF protein expression with this probiotic yeast.