title: High-yield production of hydrophobins RodA and RodB from Aspergillus fumigatus in Pichia pastoris.
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.
authors.affiliation: Center for Microbial Biotechnology, Technical University of Denmark, DTU Systems Biology, Søltofts plads 221, Kgs. Lyngby, Denmark. mohp@bio.dtu.dk
authors.forename: Mona Højgaard
authors.initials: MH
authors.lastname: Pedersen
authors.forename: Irina
authors.initials: I
authors.lastname: Borodina
authors.forename: Jacob Lange
authors.initials: JL
authors.lastname: Moresco
authors.forename: Winnie Edith
authors.initials: WE
authors.lastname: Svendsen
authors.forename: Jens Christian
authors.initials: JC
authors.lastname: Frisvad
authors.forename: Ib
authors.initials: I
authors.lastname: Søndergaard
mesh_terms: D001232:Aspergillus fumigatus / Q000235:genetics*; D001709:Biotechnology / Q000379:methods; D005285:Fermentation; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins