High-yield production of hydrophobins RodA and RodB from <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis> |
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Authors: | Mona Højgaard Pedersen Irina Borodina Jacob Lange Moresco Winnie Edith Svendsen Jens Christian Frisvad Ib Søndergaard |
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Institution: | 1.Center for Microbial Biotechnology,Technical University of Denmark, DTU Systems Biology,Kgs. Lyngby,Denmark;2.DTU Nanotech,Technical University of Denmark,Kgs. Lyngby,Denmark |
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Abstract: | Hydrophobins are small fungal proteins with amphipatic properties and the ability to self-assemble on a hydrophobic/hydrophilic
interface; thus, many technical applications for hydrophobins have been suggested. The pathogenic fungus Aspergillus fumigatus expresses the hydrophobins RodA and RodB on the surface of its conidia. RodA is known to be of importance to the pathogenesis
of the fungus, while the biological role of RodB is currently unknown. Here, we report the successful expression of both hydrophobins
in Pichia pastoris and present fed-batch fermentation yields of 200–300 mg/l fermentation broth. Protein bands of expected sizes were detected
by SDS-PAGE and western blotting, and the identity was further confirmed by tandem mass spectrometry. Both proteins were purified
using his-affinity chromatography, and the high level of purity was verified by silver-stained SDS-PAGE. Recombinant RodA
as well as rRodB were able to convert a glass surface from hydrophilic to hydrophobic similar to native RodA, but only rRodB
was able to decrease the hydrophobicity of a Teflon-like surface to the same extent as native RodA, while rRodA showed this
ability to a lesser extent. Recombinant RodA and native RodA showed a similar ability to emulsify air in water, while recombinant
RodB could also emulsify oil in water better than the control protein bovine serum albumin (BSA). This is to our knowledge
the first successful expression of hydrophobins from A. fumigatus in a eukaryote host, which makes it possible to further characterize both hydrophobins. Furthermore, the expression strategy
and fed-batch production using P. pastoris may be transferred to hydrophobins from other species. |
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