Overproduction and initial characterization of a class II hydrophobin fusion protein heterologously produced in Pichia pastoris

Hydrophobins are a class of small amphiphilic proteins secreted by filamentous fungi that play many roles in fungal development and morphogenesis. Due to their interfacial self-assembly and capabilities as biosurfactants, these proteins have been a focus of research that aims to understand their structure-function relationships and harness their biosurfactant properties for specific applications. In this thesis, I use the methylotrophic yeast Pichia pastoris as a eukaryotic production host for inducible recombinant protein expression, secretion, and initial characterization of a class II hydrophobin fusion protein based on the hydrophobin HFBI from Trichoderma reesei. An immunodetection-based 96 well plate format for high-throughput screening of high-expression clones was successfully implemented. Induction and expression conditions were optimized, with the optimal induction occurring at OD600 = 1.0 in unbuffered complex methanol media. Recombinant hydrophobin was successfully overexpressed, secreted, and purified by ion affinity chromatography, producing a pure product at the expected molecular weight. Multimerization of purified hydrophobin was analyzed by tricine-SDS-PAGE and dynamic light scattering under reducing and nonreducing conditions. Multimer bands were only seen in unreduced samples with PAGE, and dynamic light scattering radii changed to approximately monomer sizes after reducing the sample disulfides. Tensiometry and ANS fluorescence were used to measure the critical aggregation concentration of the purified hydrophobin. Circular dichroism spectroscopy showed that hydrophobin is thermally stable, even after heating to 90°C.