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) |
|---|---|
| Pages (from-to) | 203-209 |
| Number of pages | 7 |
| Journal | Protein Expression and Purification |
| Volume | 58 |
| Issue number | 2 |
| DOIs | |
| State | Published - Apr 2008 |
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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 journal › Article
}
TY - JOUR
T1 - Expression and purification of cysteine introduced recombinant saporin
AU - Günhan, Emine
AU - Swe, Mimi
AU - Palazoglu, Mine
AU - Voss, John C
AU - Chalupa, Leo M.
PY - 2008/4
Y1 - 2008/4
N2 - 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.
AB - 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.
KW - Immunotoxin
KW - Saporin
UR - http://www.scopus.com/inward/record.url?scp=39549084730&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=39549084730&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2007.11.005
DO - 10.1016/j.pep.2007.11.005
M3 - Article
C2 - 18164211
AN - SCOPUS:39549084730
VL - 58
SP - 203
EP - 209
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
IS - 2
ER -