Expression, purification, and refolding of diverse class IB hydrophobins

Hydrophobins are low molecular weight proteins secreted by fungi that are extremely surface-active and able to self-assemble into larger structures. Due to their unusual biochemical properties, hydrophobins are an attractive target for commercial applications such as drug emulsification and surface modification. When produced in E. coli, hydrophobins are often not soluble and need to be refolded. In this work we use SHuffle T7 Express E. coli coupled with glutathione redox buffers to produce and refold four distinct class IB hydrophobins that originate from Phanerochaete carnosa (PC1), Wallemia ichthyophaga (WI1), Serpula lacrymans (SL1), and Schizophyllum commune (SC16). Proper refolding and function of these purified hydrophobins was confirmed using nuclear magnetic resonance spectroscopy and thioflavin T assays. These results indicate that class IB hydrophobins can be consistently produced and purified from E. coli, aiding future structural and biochemical studies that require highly pure hydrophobins.