Back
 ABC  Vol.7 No.2 , April 2017
Development and Applications of a Calmodulin-Based Fusion Protein System for the Expression and Purification of WW and Zinc Finger Modules
Abstract: 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.
Cite this paper: 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.
References

[1]   Chant, A., Kraemer-Pecore, C.M., Watkin, R. and Kneale, G.G. (2005) Attachment of a Histidine Tag to the Minimal Zinc Finger Protein of the Aspergillus nidulans Gene Regulatory Protein Area Causes a Conformational Change at the DNA-Binding Site. Protein Expression and Purification, 39, 152-159.

[2]   Ababou, A., Shenvi, R.A. and Desjarlais, J.R. (2001) Long-Range Effects on Calcium Binding and Conformational Change in the N-Domain of Calmodulin. Biochemistry, 40, 12719-12726.

[3]   Macias, M.J., Hyvonen, M., Baraldi, E., Schultz, J., Sudol, M., Saraste, M. and Oschkinat, H. (1996) Structure of the WW Domain of a Kinase-Associated Protein Complexed with a Proline-Rich Peptide. Nature, 382, 646-649.
https://doi.org/10.1038/382646a0

[4]   Kraemer-Pecore, C.M., Lecomte, J.T. and Desjarlais, J.R. (2003) A de Novo Redesign of the WW Domain. Protein Science, 12, 2194-2205.
https://doi.org/10.1110/ps.03190903

[5]   Desai, U.A., Sur, G., Daunert, S., Babbitt, R. and Li, Q. (2002) Expression and Affinity Purification of Recombinant Proteins from Plants. Protein Expression and Purification, 25, 195-202.

[6]   Li, C.Q., Ye, P., Cao, Z., Wang, H., Lu, L., Nicastro, P., Wood, E., Robert, J.J., Ouwehand, W.H., Hill, F., Lopez, J.A. and Wardell, M.R. (2001) Expression of the Amino-Terminal Domain of Platelet Glycoprotein Ibα: Exploitation of a Calmodulin Tag for Determination of Its Functional Activity. Protein Expression and Purification, 22, 200-210.

[7]   Schauer-Vukasinovic, V. and Daunert, S. (1999) Purification of Recombinant Proteins Based on the Interaction between a Phenothiazine-Derivatized Column and a Calmodulin Fusion Tail. Biotechnology Progress, 15, 513-516.
https://doi.org/10.1021/bp990058l

[8]   Hristova, M., Veith, C., Habibovic, A., Lam, Y.W., Deng, B., Geiszt, M., Janssen-Heininger, Y.M. and van der Vliet, A. (2014) Identification of Duox1-Dependent Redox Signaling through Protein S-Glutathionylation in Airway Epithelial Cells. Redox Biology, 2, 436-46.
https://doi.org/10.1016/j.redox.2013.12.030

[9]   Pecore, C.M.K. (2002) Computational Design and Experimental Characterization of Protein Domains. The Pennsylvania State University, State College.

[10]   Ababou, A. and Desjarlais, J.R. (2001) Solvation Energetics and Conformational Change in EF-Hand Proteins. Protein Science, 10, 301-312.
https://doi.org/10.1110/ps.33601

 
 
Top