Hydrophobins are a family of cysteine-rich proteins (7–9 kDa) unique to filamentous fungi. They exist in a soluble form and also self-assemble into organised amphipathic monolayers once encountering hydrophilic: hydrophobic interfaces. Hydrophobins play many roles in fungal biology, including lowering the surface tension of the growth medium and allowing aerial hyphae to breach the meniscus. The coating of hyphae and spores with hydrophobins also prevents them from wetting and desiccation. Hydrophobins are therefore of biotechnological interest as they are biocompatible surfactants capable of coating surfaces and changing surface wettability. They are also known to evade the immune response in humans. Potential applications range from coating medical implants, use as surfactants and in drug delivery.  

Hydrophobins are characterised by four intramolecular disulphide bonds critical to their structure and function. As such, recombinant production of hydrophobins requires complex methods for oxidative refolding. In addition, an expression system that allows for multiple hydrophobin mutants to be produced and screened simultaneously is highly desirable. The cell free (CF) protein synthesis system is an in vitro method of protein production that has been demonstrated to produce high yields of fully folded protein with multiple disulphide bonds while allowing for rapid screening of protein structure using labelled amino acids.

I will present the progress being made in the optimisation of the CF system in the production of hydrophobins. The conditions that were optimised include: the concentrations and types of reducing agent and pH range. Our results demonstrate that folded and functional hydrophobins can be produced using the CF system in sufficient quantities for structural studies and functional screens. Future work would incorporate isotopically labelled and/or non-natural amino acids into hydrophobins to aid the characterisation, functionalization and design of hydrophobin variants.