Cell-free Synthesis of Correctly Folded Proteins with Multiple Disulphide Bonds: Production of Fungal Hydrophobins.

Cell-free synthesis is a powerful technique that uses the transcriptional and translational machinery extracted from cells to create proteins without the constraints of living cells. Here, we report a cell-free protein production protocol using Escherichia coli lysate (Figure 1) to successfully express a class of proteins (known as hydrophobins) with multiple intramolecular disulphide bonds which are typically difficult to express in a soluble and folded state in the reducing environments found inside a cell. In some cases, the inclusion of a recombinant disulphide isomerase DsbC further enhances the expression levels of correctly folded hydrophobins. Using this protocol, we can achieve milligram levels of protein expression per ml of reaction. While our target proteins are the fungal hydrophobins, it is likely that this protocol with some minor variations can be used to express other proteins with multiple intramolecular disulphide bonds in a natively folded state. Graphic abstract: Figure 1.Workflow for cell-free protein expression and single-step purification using affinity chromatography. A. E. coli S30 lysate prepared as described in Apponyi et al. (2008) can be stored for up to several years at -80°C without any loss of activity in our experience. B. The S30 lysate, plasmid DNA that encodes for the protein of interest along with an affinity tag and components required for transcription and translation are added to the reaction mix. Following a single-step protein purification, the protein of interest can be isolated for further use.