Introduction: Hydrophobins are small amphipatic proteins present on the spore surface of filamentous fungi. They most likely play an important role in the attachment of spores to a solid phase. The pathogenic fungus Aspergillus fumigatus expresses the hydrophobins RodA and RodB on the surface of its conidia and these may be of importance to the pathogenesis of the fungus. Although heterologous expression of hydrophobins has proven to be a challenge by past investigators, we made it the aim of this project to produce pure hydrophobins in sufficient quantities for further characterication and investigation using the expression host Pichia pastoris. Methods and materials: The genes encoding hydrophobins RodA and RodB were amplified by RT-PCR with gene-specific primers from the total RNA isolated from the spores of A. fumigatus (AF296 strain). The resulting cDNA was cloned into TOPO vectors using TOPO TA Cloning (Invitrogen), and the inserts were sequenced. The genes were further amplified by PCR to generate overhangs with specific restriction sites and cloned into expression vectors pPICZA and pPICZB while adding a 6xHis-tag to the C-terminal of both hydrophobins. The pPICZA vector expresses proteins with the signal sequence of alfa-mating factor from Saccharomyces cerevisia known to work well for protein secretion from P. pastoris and the pPICZB plasmids had proteins cloned with their native signal sequences. The plasmids were linearized, transformed into P. pastoris strain X33 and transformants were selected by zeocin resistance. The presence of the RodA and RodB genes in the transformants was confirmed by colony PCR. The expression of RodA and RodB genes was induced by growing cells in culture flasks for three days in buffered complex methanol medium as protein production was dependent on the methanol-induced AOX1 promoter. The protein production was analyzed by SDS-PAGE, coomassie and silver-stained, as well as western blotting using a detection antibody (Penta-His HRP conjugate, Qiagen). Recombinant RodA and RodB were purified using His-select Nickel Affinity gel (Sigma-Aldrich, Saint Louis, MO, USA). Results: P. pastoris cultures expressing hydrophobins resulted in increased foaming, which was attributed to the presence of secreted hydrophobins. Protein bands of expected size were detected in both the fermentation broth and the foam by SDS-PAGE and western blotting. Optimization of the purification of hydrophobins and functional investigations are being carried out at the moment. Conclusion: Hydrophobins RodA and RodB from Aspergillus fumigatus were successfully expressed and secreted by yeast host Pichia pastoris.
|Publication status||Published - 2009|
|Event||International Conference on Environmental, Industrial and Applied Microbiology : BioMicroWorld 2009 - Lissabon, Portugal|
Duration: 1 Jan 2009 → …
|Conference||International Conference on Environmental, Industrial and Applied Microbiology : BioMicroWorld 2009|
|Period||01/01/2009 → …|