title: A novel fusion protein diphtheria toxin-stem cell factor (DT-SCF)-purification and characterization.
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.
authors.affiliation: Department of Biotechnology, Stem Cell and Molecular Biology Laboratory, Indian Institute of Technology Madras, Bhupat and Jyoti Mehta School of Biosciences, Chennai 600036, India.
authors.forename: Sirisha
authors.initials: S
authors.lastname: Potala
authors.forename: Rama Shanker
authors.initials: RS
authors.lastname: Verma
mesh_terms: D001709:Biotechnology / Q000379:methods*; D002453:Cell Cycle; D002460:Cell Line; D003001:Cloning, Molecular; D004167:Diphtheria Toxin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D019089:Stem Cell Factor / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D004167:Diphtheria Toxin; D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor; D019009:Proto-Oncogene Proteins c-kit