Protein Purification by Ethanol-Induced Phase Transitions of the Elastin-like Polypeptide (ELP)

Thermoresponsive elastin-like polypeptide (ELP) tags provide an efficient and highly scalable route to purifying recombinantly expressed proteins without the need for chromatography. By changing temperature and/or salt content, ELPs can be selectively precipitated and resolubilized, which enables the purification of the fused target protein. Despite its advantages, the method often requires addition of molar level salts for precipitation, making it difficult to control the final product salinity. Control over product salinity is especially important when fabricating protein-based materials, such as hydrogels, which often contain protein concentrations >= 10 wt % in their final state. One way to circumvent the issue of high-salt protein solutions is to take advantage of ELP cononsolvency in water/ethanol solvents. This work investigates ethanol-induced ELP precipitations as a route to purify and desalt ELP-tagged proteins. Superfolder green fluorescent protein (sfGFP) tagged with an ELP (ELP-sfGFP) was used as a model protein to develop this ethanol-based purification process. A final protein product with high purity and low salinity was obtained after one cycle of sodium chloride (NaCl)-induced precipitation followed by one cycle of ethanol-induced precipitation. ELP-sfGFP purified using this method was found to have 0.9 +/- 0.4 mM and 1.48 +/- 0.05 mM equivalents of NaCl when purified from liter-scale and well-plate scale expressions, respectively. Achieving low salinities from well-plate expression is particularly useful as this format does not permit use of other desalting techniques such as dialysis. However, the procedure is only viable for proteins that are robust to incubation in moderate concentrations of ethanol. The method's applicability in generating protein materials was demonstrated by the well-plate scale purification and desalting of an ELP-based protein hydrogel. This new process for selectively precipitating and desalting ELP-tagged proteins will enable high-throughput screening of protein materials that require tight control of buffer conditions.