Expression and purification of cysteine introduced recombinant saporin

Emine Günhan, Mimi Swe, Mine Palazoglu, John C Voss, Leo M. Chalupa

Research output: Contribution to journalArticle

4 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)203-209
Number of pages7
JournalProtein Expression and Purification
Volume58
Issue number2
DOIs
StatePublished - Apr 2008

Fingerprint

Purification
Cysteine
Isomers
Toxicity
Ribosome Inactivating Proteins
Immunotoxins
saporin
Ion Exchange Chromatography
Chromatography
Fluorescent Dyes
Labeling
Inhibitory Concentration 50
Assays
Ion exchange
Proteins
Immunoglobulin G
Mutation
Antibodies
DNA

Keywords

  • Immunotoxin
  • Saporin

ASJC Scopus subject areas

  • Biochemistry

Cite this

Expression and purification of cysteine introduced recombinant saporin. / Günhan, Emine; Swe, Mimi; Palazoglu, Mine; Voss, John C; Chalupa, Leo M.

In: Protein Expression and Purification, Vol. 58, No. 2, 04.2008, p. 203-209.

Research output: Contribution to journalArticle

Günhan, Emine ; Swe, Mimi ; Palazoglu, Mine ; Voss, John C ; Chalupa, Leo M. / Expression and purification of cysteine introduced recombinant saporin. In: Protein Expression and Purification. 2008 ; Vol. 58, No. 2. pp. 203-209.
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