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.
Original language | English (US) |
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Pages (from-to) | 203-209 |
Number of pages | 7 |
Journal | Protein Expression and Purification |
Volume | 58 |
Issue number | 2 |
DOIs | |
State | Published - Apr 2008 |
Keywords
- Immunotoxin
- Saporin
ASJC Scopus subject areas
- Biochemistry