- Brian Sipko, Chemical Engineering, University of Delaware
- Mark Blenner, Chemical and Biomolecular Engineering, University of Delaware
Hydrophobins are small, cysteine-rich proteins, native to a variety of filamentous fungi species. Driven by the hydrophobic effect, hydrophobins have a unique ability to self-assemble at interfaces, thus creating a modified surface. These surface modifications have a role in a variety of different applications, ranging from plastic degradation, to medical device enhancement. Extraction of the hydrophobins directly from the fungal species is not an efficient, or even entirely practical, method. Beyond this, all hydrophobins have differing solubility; this divides them into Class I and II, where Class I is only soluble with two reagents (Trifluoroacetic acid, formic acid) and Class II is soluble with a much larger variety of solvents. Because of this, much more work has been done on Class II hydrophobins.
My research has focused on using recombination to express RodA, a Class I hydrophobin native to the fungus Aspergillus fumigatus. The yeast strain Yarrowia lipolytica is used as a host for expression, due to its well-developed engineering tools, high substrate compatibility, and native signal peptides. To isolate recombinant RodA, protein purification and detection methods have been pursued. The protein was designed with a 6xHis-tag which allows for purification using Fast Protein Liquid Chromatography (FPLC) and detection by western blotting. Purification and detection via these methods proved unsuccessful, but provided insight towards alternate approaches, including: Trifluoroacetic acid solubilization, methanol-chloroform precipitation, and pull-down assays. These efforts have yielded much more promising results than the methods involving the 6xHis-tag. Future work will include improving the solubility of RodA by introducing a Small Ubiquitin-like modifying protein (SUMO) tag, and exploring the effects of various signal peptides on RodA secretion in Y. lipolytica.