title: Hydrophobin-Based Surface Engineering for Sensitive and Robust Quantification of Yeast Pheromones.
abstract: Detection and quantification of small peptides, such as yeast pheromones, are often challenging. We developed a highly sensitive and robust affinity-assay for the quantification of the α-factor pheromone of Saccharomyces cerevisiae based on recombinant hydrophobins. These small, amphipathic proteins self-assemble into highly stable monolayers at hydrophilic-hydrophobic interfaces. Upon functionalization of solid supports with a combination of hydrophobins either lacking or exposing the α-factor, pheromone-specific antibodies were bound to the surface. Increasing concentrations of the pheromone competitively detached the antibodies, thus allowing for quantification of the pheromone. By adjusting the percentage of pheromone-exposing hydrophobins, the sensitivity of the assay could be precisely predefined. The assay proved to be highly robust against changes in sample matrix composition. Due to the high stability of hydrophobin layers, the functionalized surfaces could be repeatedly used without affecting the sensitivity. Furthermore, by using an inverse setup, the sensitivity was increased by three orders of magnitude, yielding a novel kind of biosensor for the yeast pheromone with the lowest limit of detection reported so far. This assay was applied to study the pheromone secretion of diverse yeast strains including a whole-cell biosensor strain of Schizosaccharomyces pombe modulating α-factor secretion in response to an environmental signal.
authors.affiliation: Institute of Genetics, Technische Universität Dresden, 01062 Dresden, Germany. Stefan.Hennig1@tu-dresden.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Hennig
authors.affiliation: Institute of Genetics, Technische Universität Dresden, 01062 Dresden, Germany. Gerhard.Roedel@tu-dresden.de.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Rödel
authors.affiliation: Institute of Genetics, Technische Universität Dresden, 01062 Dresden, Germany. Kai.Ostermann@tu-dresden.de.
authors.forename: Kai
authors.initials: K
authors.lastname: Ostermann
mesh_terms: D015374:Biosensing Techniques; D057927:Hydrophobic and Hydrophilic Interactions; D010455:Peptides; D010675:Pheromones; D011506:Proteins; D012441:Saccharomyces cerevisiae
chemical_list: D010455:Peptides; D010675:Pheromones; D011506:Proteins
keywords: analyte detection; biosensor; hydrophobin; surface functionalization; whole-cell biosensor; yeast pheromone