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A Novel Fusion Protein Diphtheria Toxin–Stem Cell Factor (DT-SCF)—Purification and Characterization

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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.

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Abbreviations

DT:

diphtheria toxin

SCF:

stem cell factor

CTAB:

cetyl trimethylammonium bromide

IPTG:

isopropyl-β-D-thiogalacto-pyranoside

TMB:

tetramethyl benzidine

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

TBS:

tris buffer saline

TBST:

tris buffer saline tween-20

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Authors and Affiliations

  1. Department of Biotechnology, Stem Cell and Molecular Biology Laboratory, Indian Institute of Technology Madras, #201 Bhupat and Jyoti Mehta School of Biosciences, Chennai, 600036, India

    Sirisha Potala & Rama Shanker Verma

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  1. Sirisha Potala
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  2. Rama Shanker Verma
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Correspondence to Rama Shanker Verma.

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Potala, S., Verma, R.S. A Novel Fusion Protein Diphtheria Toxin–Stem Cell Factor (DT-SCF)—Purification and Characterization. Appl Biochem Biotechnol 162, 1258–1269 (2010). https://doi.org/10.1007/s12010-009-8896-1

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  • DOI: https://doi.org/10.1007/s12010-009-8896-1

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