Expression and purification of cysteine introduced recombinant saporin.
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 IC(50) 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.
Günhan
Emine
E
Department of Neurobiology, Physiology, and Behavior, University of California, One Shields Avenue, 196 Briggs Hall, Davis, CA 95616, USA. egunhan@ucdavis.edu
Swe
Mimi
M
Palazoglu
Mine
M
Voss
John C
JC
Chalupa
Leo M
LM
D000818:Animals; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D000922:Immunotoxins / Q000494:pharmacology; D008301:Maleimides / Q000737:chemistry; D051379:Mice; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D011817:Rabbits; D054789:Ribosome Inactivating Proteins, Type 1 / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D012270:Ribosomes / Q000187:drug effects; D000076985:Saporins
D007074:Immunoglobulin G; D000922:Immunotoxins; D008301:Maleimides; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins; D003545:Cysteine