Author(s)

Christopher G. Toomey¹, David Weiss¹, Alan Chant², Megan Ackerman¹, Bethany A. Ahlers^3,4, Ying-Wai Lam^3,4, Christopher Ricciardi¹, Dana Bourne⁵, Christina M. Kraemer-Chant^1*

¹Department of Chemistry, Saint Michael’s College, Colchester, VT, USA.
²Department of Chemistry, University of Vermont, Burlington, VT, USA.
³Department of Biology, University of Vermont, Burlington, VT, USA.
⁴Vermont Genetics Network Proteomics Facility, University of Vermont, Burlington, VT, USA.
⁵Department of Biochemistry, University of Vermont, Burlington, VT, USA.

Calmodulin from Homo sapiens is an α-helical calcium-binding protein that expresses to high levels in E. coli. When the N-terminus of a calmodulin variant is bound to Ca²⁺, it undergoes a conformational change, exposing hydrophobic pockets. This property can be utilized for purification purposes, as these pockets bind to phenyl sepharose resin with high affinity. Washing with EDTA chelates the Ca²⁺ ions from the protein, inducing a conformational change back to the more folded state and eluting the protein from the column. We describe herein the use of a protein expression and purification technique using the calmodulin variant and a short linker for proteolytic cleavage by the mutant NIa-Pro tobacco etch virus protease. We have shown this approach to be useful in obtaining purified quantities of various small proteins that could not be expressed using other methods, including high enough concentrations of a designed WW domain protein for NMR structural analysis. We have also obtained promising results on the usefulness of this procedure to express and purify zinc finger proteins without the addition of zinc ions or other cofactors.

Biomaterials, Protein Purification, Expression Systems, Protein Expression

Toomey, C. , Weiss, D. , Chant, A. , Ackerman, M. , Ahlers, B. , Lam, Y. , Ricciardi, C. , Bourne, D. and Kraemer-Chant, C. (2017) Development and Applications of a Calmodulin-Based Fusion Protein System for the Expression and Purification of WW and Zinc Finger Modules. Advances in Biological Chemistry, 7, 89-106. doi: 10.4236/abc.2017.72006.