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A novel fusion protein diphtheria toxin-stem cell factor (DT-SCF)-purification and characterization
Published in
2010
PMID: 20084469
Volume: 162
   
Issue: 5
Pages: 1258 - 1269
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. © 2010 Springer Science+Business Media, LLC.
About the journal
JournalApplied Biochemistry and Biotechnology
ISSN02732289
Open AccessNo
Concepts (53)
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    Cell cycle analysis
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    Diphtheria toxin
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    IMMUNOTOXINS
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    Protein expression
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    Refolding
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    SCF
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    AFFINITY CHROMATOGRAPHY
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    Amino acids
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    Diseases
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    Gene expression
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    Organic acids
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    Proteins
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    Purification
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    Toxic materials
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    Stem cells
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    DIPHTHERIA TOXIN STEM CELL FACTOR
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    Genomic dna
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    POLYHISTIDINE TAG
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    Stem cell factor
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    Unclassified drug
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    ACUTE LYMPHOBLASTIC LEUKEMIA
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    Animal cell
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    Article
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    Cancer cell culture
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    CELL CYCLE PARAMETERS
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    Cell viability
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    Chemical structure
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    Chronic myeloid leukemia
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    Concentration (parameters)
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    Corynebacterium diphtheriae
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    Cytotoxicity test
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    DNA EXTRACTION
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    Dna sequence
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    Genetic code
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    Human
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    Human cell
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    IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY
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    Molecular cloning
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    Nonhuman
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    PANCREAS CARCINOMA
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    Protein analysis
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    Protein purification
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    Sensitivity and specificity
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    Uterine cervix carcinoma
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    Biotechnology
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    Cell cycle
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    Cell line
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    Cloning, molecular
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    Fluorescent antibody technique
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    Humans
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    IMMUNOBLOTTING
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    Proto-oncogene proteins c-kit
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    Recombinant fusion proteins