Expression and purification of cysteine introduced recombinant saporin

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 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.

Figure 1. SDS-PAGE (A) and Western Blot (B) Analysis of saporin, sap-3 isomer and Cys255sap-3

A, SDS-PAGE analysis was done with non reducing SDS gels. Lane 1: protein marker, lane 2: commercial saporin, lane 3 and 4 Cys255sap-3, lane 5: sap-3 isomer. B, Western Blot Assay using the polyclonal anti-saporin antibodies (dilution 0.36 μg/ml). Lane 1: protein marker, lane2: commercial saporin, lane 3: sap-3 isomer, lanes 4 and 5: Cys255sap-3.

Figure 2. SDS-PAGE Analysis of Alexa 568 tagged Cys255sap-3. Left column

(lanes 1 and 2), image of non reducing SDS-PAGE gel scanned on Typhoon Scanner using the 580 nm laser. Right column (lanes 3 and 4) Coomassie stained non reducing SDS-PAGE gel (the same gel as left column). Lanes 1 and 4 protein marker, lanes 2 and 3: Alexa 568 tagged Cys255Sap-3, (2 μg).

Figure 3. SDS-PAGE Analysis of recombinant saporin, mouse anti human IgG and recombinant saporin conjugated to mouse anti-human IgG

Lane 1, protein marker, lane 2, Cys255sap-3 (2 μg), lane 3, mouse anti human IgG (2.4 μg), lane 4, Cys255sap-3 conjugated to mouse anti human IgG (3.8 μg).

Figure 4. Semi-log graph showing the protein synthesis inhibition as a function of toxin concentration

Activities for Cys255sap-3, Sap-3 isomer and commercial saporin were tested by protein synthesis inhibition assay. 1 μl mRNA was added to 0.5 ml RNase-free microcentrifuge tubes and incubated 3 minutes at 65°C. 1 μl of samples (dilutions of Cys255sap-3, Sap-3 isomer and commercial saporin), 7 μl of rabbit reticulocyte lysate (Promega), 1 μl of amino acid minus mixture and 0.2 μl of RNasin were added to respective tubes and incubated at 30°C in a waterbath for 30 minutes. 2 μl of samples from each tube were added on 96-well opaque plate and read on L-Max luminometer using 25 μl Luciferase Assay Reagent (Promega). Data were analyzed using MatLab. Green; Sap-3 isomer, Blue; Cys255sap-3, Red; commercial saporin (purchased from Advanced Targeting Systems).