Genetic Alterations Which Facilitate Protein Purification: Applications in the Biopharmaceutical Industry
A large number of genetic modifications have been employed to facilitate the purification of numerous recombinant proteins. These techniques have been used for both commercial and research purposes and in a variety of different expression systems. This paper will explore the value of this approach as a research tool and focus primarily on two methods. The first method utilizes a metal-chelate fusion and the second an immuno-affinity fusion. These two methods can be successfully employed in virtually any expression system. The resulting purified protein can be used for a variety of research purposes including refolding studies and pre-clinical biology.
KeywordsRecombinant Protein Genetic Engineering Technique Refold Condition Metal Chelate Chromatography Enterokinase Cleavage Site
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- 1.S. J. Brewer, B. L Haymore, T. P. Hopp and H. M. Sassenfeld, Engineering Proteins to Enable Their Isolation in a biologically active form. In: Purification and Analysis of Recombinant Proteins. R. Seetharam, S. K. Sharma eds. Marcel Dekker, Inc., NY (1991) pp. 239–266.Google Scholar
- 2.M. Uhlen and T. Moks, Gene fusions for purpose of expression: An Introduction. In: Methods in Enzymology. R. Williamson, ed. Academic Press, NY (1981) pp. 129–143.Google Scholar
- 7.H. M. Sassenfeld, Engineering proteins for purification: Use of a C-terminal polyarginine fusion. PhD Thesis, University of Liverpool, UK.Google Scholar
- 8.A. J. Brake, J. P. Merryweather, D. G. Coit, U. A. Heberlein, F. R. Masiarz, G. T. Mullenbach, M. S. Urdea, P. Valenzuela, and P. J. Barr, α-Factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 81:4642 (1984).PubMedCrossRefGoogle Scholar
- 10.S. S. Suh, B. L Haymore, and F. H. Arnold, Characterization of His-X3-His sites in α-helices of synthetic metal-binding bovine somatotropin. Proteins in Engineering, in press.Google Scholar
- 12.E. S. Hemdan, Y. Zhao, E. Sulkowski, and J. Porath, Surface topography of histidine residues: A facile probe by immobilized metal ion affinity chromatography. Proc. Natl. Acad. Sci. USA 86:1811–1815.Google Scholar
- 14.B. L Haymore, G. S. Bild, S. S. Abdel-Meguid, M. C. Clare, G. G. Krivi, W. J. Salsgiver, and N. R. Staten, Introducing strong metal-binding sites into somatotropin. Facile and efficient metal-affinity purification, Submitted.Google Scholar