Related Experiment Video
Updated: Jun 17, 2026
Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)
Published on: September 17, 2019
A novel fusion protein diphtheria toxin-stem cell factor (DT-SCF)-purification and characterization
Sirisha Potala1, Rama Shanker Verma
1Department of Biotechnology, Stem Cell and Molecular Biology Laboratory, Indian Institute of Technology Madras, Bhupat and Jyoti Mehta School of Biosciences, Chennai 600036, India.
Abstract:
Fusion toxins are an emerging class of targeted therapeutics for the treatment of cancer. Diphtheria toxin-stem cell factor (DT-SCF) is one such novel fusion toxin designed to target malignancies expressing c-kit. Since, c-kit overexpression has been reported on many types of cancers, it appeared to be a reasonably good molecule to target. In the present study, we report construction, expression, purification, and characterization of DT-SCF. DT-SCF gene coding for 1-387 amino acids of diphtheria toxin, His-Ala linker, 2-141 amino acids of SCF was cloned into expression vector with C terminal His tag. The induced DT-SCF protein was exclusively expressed in insoluble fraction. Purification of DT-SCF was achieved by inclusion body isolation and metal affinity chromatography under denaturing and reducing conditions. Purified DT-SCF was renatured partially on-column by gradually reducing denaturant concentration followed by complete refolding through rapid dilution technique. Cell viability assay provided the evidence that DT-SCF is a potent cytotoxic agent selective to cells expressing c-kit. The novelty of this study lies in employing SCF as a ligand in construction of fusion toxin to target wide range of malignancies expressing c-kit. Efficacy of DT-SCF fusion toxin was demonstrated over a range of malignancies such as chronic myeloid leukemia (K562), acute lymphoblastic leukemia (MOLT4), pancreatic carcinoma (PANC-1), and cervical carcinoma (HeLa 229). This is the first study reporting specificity and efficacy of DT-SCF against tumor cells expressing c-kit. There was significant correlation (P = 0.007) between c-kit expression on cells and their sensitivity to DT-SCF fusion toxin.
Insights
This study developed a novel fusion toxin, diphtheria toxin-stem cell factor (DT-SCF), to target cancers overexpressing c-kit. DT-SCF demonstrated potent and selective cytotoxicity against various cancer cells, highlighting its therapeutic potential.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Fusion toxins represent an emerging class of targeted cancer therapeutics.
- Overexpression of c-kit is observed in various malignancies, making it a viable therapeutic target.
Purpose of the Study:
- To construct, express, purify, and characterize a novel fusion toxin, diphtheria toxin-stem cell factor (DT-SCF).
- To evaluate the specificity and efficacy of DT-SCF against c-kit expressing tumor cells.
Main Methods:
- DT-SCF gene encoding diphtheria toxin and stem cell factor was cloned into an expression vector.
- Protein expression, purification via metal affinity chromatography, and renaturation techniques were employed.
- Cell viability assays were conducted to assess cytotoxicity and selectivity.
Main Results:
- DT-SCF was successfully expressed and purified under denaturing conditions, followed by refolding.
- Cell viability assays confirmed DT-SCF as a potent cytotoxic agent selective for c-kit expressing cells.
- Significant correlation (P = 0.007) was observed between c-kit expression levels and DT-SCF sensitivity across various cancer types.
Conclusions:
- DT-SCF is a novel and effective fusion toxin targeting malignancies expressing c-kit.
- The study demonstrates the specificity and efficacy of DT-SCF against chronic myeloid leukemia, acute lymphoblastic leukemia, pancreatic carcinoma, and cervical carcinoma cell lines.
- DT-SCF shows promise as a targeted therapeutic agent for a wide range of c-kit-positive cancers.
