Calmodulin from Homo sapiens is an α-helical calcium binding protein that expresses to high levels in E. coli. When calmodulin binds to calcium ions, it undergoes a conformational change, resulting in the exposure of hydrophobic pockets. Due to this increase in hydrophobic surface area, the protein binds with greatly increased affinity to phenyl sepharose, a hydrophobic resin, when in the presence of Ca2+. It can be eluted from the resin through the addition of EDTA. We have shown that calmodulin’s affinity to phenyl sepharose can be used as part of a fusion system for the purification of other proteins. For this project, the N-terminus of a mutated calmodulin protein (N-Cam.Y) was used as the N-terminus of the fusion system. A proteolytic cleavage site was located after N-Cam.Y, followed by the protein of interest. The cleavage site was added so that the calmodulin portion of the system could be removed after purification of the fusion system. We developed this system for expression in E. coli and purification using phenyl sepharose in the presence of calcium ions. The calcium ions were then chelated from the calmodulin portion of the fusion system using an EDTA buffer, which resulted in significantly decreased affinity of the N-Cam.Y to the phenyl sepharose and concomitant washing of the system from the column. Resultant fusion protein can then be cleaved and the target protein can be further purified using other methods. This method has proven effective for the purification of WW domains, a small beta-sheet protein that could not be purified with other commercially-available purification methods such as His tags. We have recently demonstrated the usefulness of this method in expressing and purifying a zinc-finger module without the addition of zinc ions. Because of this module’s size, adding a six-His tag for purification could potentially alter the fold or function of the target protein. The goal of our research is to determine whether our fusion system is useful for the expression of many different hard-to-express proteins. In short, we wish to develop a system for protein purification that is straightforward and will not alter the folding or functionality of the target proteins.

Development and analysis of a novel protein fusion system for the expression and purification of proteins

253rd National Meeting (2017)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.