Title

Expression and purification of cysteine introduced recombinant saporin

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.

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