Cell-free expression of natively folded hydrophobins

Hydrophobins are a family of cysteine-rich proteins unique to filamentous fungi. The proteins are produced in a soluble form but self-assemble into organised amphipathic layers at hydrophilic:hydrophobic interfaces. These layers contribute to transitions between wet and dry environments, spore dispersal and attachment to surfaces for growth and infection. Hydrophobins are characterised by four disulphide bonds that are critical to their structure and function. Thus, obtaining correctly folded, soluble and functional hydrophobins directly from bacterial recombinant expression is challenging and in most cases, initial denaturation from inclusion bodies followed by oxidative refolding are required to obtain folded proteins. Here, we report the use of cell-free expression with E. coli cell lysate to directly obtain natively folded hydrophobins. All six of the hydrophobins tested could be expressed after optimisation of redox conditions. For some hydrophobins, the inclusion of the disulfide isomerase DsbC further enhanced expression levels. We are able to achieve a yield of up to 1 mg of natively folded hydrophobin per mL of reaction. This has allowed the confirmation of the correct folding of hydrophobins with the use of 15N-cysteine and 15N–1H nuclear magnetic resonance experiments within 24 h of starting from plasmid stocks. •Cell-free expression with E. coli cell lysate can be used to directly obtain natively folded hydrophobins.•For some hydrophobins, addition of the disulfide isomerase DsbC can further enhance hydrophobin expression levels.•After optimisation, a yield of 0.1 to 1 milligram of natively folded hydrophobins per mL of reaction can be obtained.•The use of 15N-cysteine and NMR experiments enables an assessment of correct hydrophobin folding within 24 hours.