Expression and purification of cysteine introduced recombinant saporin

Authors

Emine Günhan, University of California, Davis
Mimi Swe, University of California, Davis
Mine Palazoglu, University of California, Davis
John C. Voss, University of California, Davis
Leo M. Chalupa, University of California, Davis

Document Type

Journal Article

Publication Date

4-1-2008

Journal

Protein Expression and Purification

Volume

58

Issue

2

DOI

10.1016/j.pep.2007.11.005

Keywords

Immunotoxin; Saporin

Abstract

Saporin, a ribosome inactivating protein is widely used for immunotoxin construction. Here we describe a mutation of saporin (sap)-3 DNA by introducing a cysteine residue, followed by protein expression and purification by ion exchange chromatography. The purified Cys255sap-3, sap-3 isomer and commercially purchased saporin, were tested for toxicity using assays measuring inhibition for protein synthesis. The IC50 values showed that the toxicity of the Cys255sap-3 is equivalent to the sap-3 isomer and commercial saporin. Reactivity of Cys255sap-3 was confirmed by labeling with a thio-specific fluorescent probe as well as conjugation with a nonspecific mouse IgG. We have found that a single cysteine within saporin provides a method for antibody conjugation that ensures a uniform and reproducible modification of a saporin variant retaining high activity. © 2007 Elsevier Inc. All rights reserved.

APA Citation

Günhan, E., Swe, M., Palazoglu, M., Voss, J., & Chalupa, L. (2008). Expression and purification of cysteine introduced recombinant saporin. Protein Expression and Purification, 58 (2). http://dx.doi.org/10.1016/j.pep.2007.11.005