Expression and purification of cysteine introduced recombinant saporin
Protein Expression and Purification
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.
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