Recombinant Production And Purification of Hydrophobin SC16 From Escherichia coli And Monitoring of Its Self-Assembly...

Hydrophobins are small amphipathic proteins that self-assemble at hydrophobic-hydrophilic interfaces to form stable waterproof coatings over fungal spores. Class I hydrophobins form particularly stable "rodlets" containing ordered amyloid-like structures that coat surfaces. This unusual surface chemistry gives them potential applications in drug delivery, foam stabilization, emulsification, and surface modification. To produce hydrophobins, there is a need for comprehensive, high-yield protocols from simple expression systems. Furthermore, it is necessary to provide facile methods to characterize hydrophobin function following recombinant production and purification. Herein, we describe protocols for the production and characterization of SC16, a class IB hydrophobin produced by the fungus Schizophyllum commune, which can be recombinantly expressed in Escherichia coli and subsequently purified under non-reducing, native conditions. This involves transformation of an expression plasmid encoding SC16 fused to an N-terminal fusion protein into E. coli, induction of protein expression, cell lysis under native conditions, affinity chromatography, and protease cleavage to isolate the hydrophobin at yields of ≥5 mg. The self-assembly of SC16 can then be monitored by end-point or kinetic fluorescence-based assays using thioflavin T. Both assays involve incubation of SC16 with prolonged mixing to promote self-assembly at air-water interfaces and are monitored by fluorescence measurements. Together, these approaches provide simplified protocols for purifying SC16 and monitoring its self-assembly and may be extended to other systems involving similar protein self-assembly.