(556d) Optimization of ELP-Intein Mediated Protein Purification by Salt Substitution | AIChE

(556d) Optimization of ELP-Intein Mediated Protein Purification by Salt Substitution


Fong, B. A. - Presenter, Princeton University
Wu, W. - Presenter, Princeton University
Wood, D. W. - Presenter, The Ohio State University

Elastin-like polypeptides (ELPs) have been used with self-cleaving inteins to yield a non-chromatographic and protease-free purification system. This method is based on the selective and reversible precipitation of ELP-tagged target proteins in response to mild heating and high concentrations of salt. Selective precipitation of the ELP-bound target is driven by competition between entropic and hydrophobic forces. At low temperatures, ordered water molecules surround the unfolded ELP chain's hydrophobic groups. At higher temperatures, ELP aggregation occurs spontaneously due to the increase in the total entropy of the solution. Adding salt to the system increases the polarity between the bulk water molecules and the ELP's hydrophobic groups, increasing the energetic cost of hydrophobic hydration, which ultimately lowers the temperature at which the ELPs spontaneously aggregate (i.e. the transition temperature).

Currently, NaCl is widely used to precipitate ELPs. However, an investigation of the Hofmeister series suggested that a more kosmotropic salt may be more effective for ELP precipitation. This series ranks ions, with kosmotropes being more effective than chaotropes, in terms of their ability to precipitate proteins and their effect on bulk water structure. It was found that by using (NH4)2SO4 instead of NaCl for ELP precipitation, the transition temperature could be lowered from 37°C to room temperature, and the working concentration of salt could be cut almost four-fold. This improvement has significant industrial relevance, since high concentrations of NaCl can corrode stainless steel equipment. In addition, the reagent cost for the precipitation step would be cut in half, which would be particularly significant at larger scales. Lastly, both specific activity and yield for our purified protein were enhanced using this optimized method, and the milder purification conditions significantly increase the attractiveness of this highly general and economical method for protein purification.