Expression and purification of cysteine introduced recombinant saporin |
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Authors: | Günhan Emine Swe Mimi Palazoglu Mine Voss John C Chalupa Leo M |
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Institution: | aDepartment of Neurobiology, Physiology, and Behavior, University of California, One Shields Avenue, 196 Briggs Hall, Davis, CA 95616, USA;bDepartment of Biochemistry, University of California, Davis, CA 95616, USA |
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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. |
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Keywords: | Saporin Immunotoxin |
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