Expression, Purification, and Therapeutic Implications of Recombinant sFRP1
| Autor: | Archita Ghoshal, Siddhartha Sankar Ghosh |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Cell Survival
Octoxynol Recombinant Fusion Proteins Gene Expression Antineoplastic Agents Bioengineering Biology Applied Microbiology and Biotechnology Biochemistry Inclusion bodies law.invention HeLa FLAG-tag Affinity chromatography law Escherichia coli Humans Cloning Molecular Molecular Biology Inclusion Bodies Expression vector Wnt signaling pathway Membrane Proteins Drug Synergism Sarcosine General Medicine biology.organism_classification Molecular biology Molecular Docking Simulation Wnt Proteins Solubility Doxorubicin Structural Homology Protein Docking (molecular) MCF-7 Cells Recombinant DNA Intercellular Signaling Peptides and Proteins Female Cisplatin HeLa Cells Plasmids Biotechnology |
| Zdroj: | Applied Biochemistry and Biotechnology. 175:2087-2103 |
| ISSN: | 1559-0291 0273-2289 |
| DOI: | 10.1007/s12010-014-1354-8 |
| Popis: | Secreted frizzled-related proteins (sFRPs) constitute a family of proteins, which impede the Wnt signaling pathway. Upregulation of the Wnt cascade is one of the multiple facets of carcinogenesis. Herein, we report the expression, solubilization, purification, characterization, and anti-cell proliferative activity of a novel recombinant GST-tagged sFRP1 of human origin. sFRP1 was cloned into pGEX-4T2 bacterial expression vector, and the recombinant protein was overexpressed in Escherichia coli BL21 (DE3). It was solubilized from inclusion bodies with N-lauroylsarcosine and Triton X-100, before being purified to homogeneity using glutathione agarose affinity chromatography column. The purified protein was characterized using Western blotting, MALDI TOF-TOF, and circular dichroism spectroscopy analysis. Homology modeling and docking studies revealed that tagging GST with sFRP1 does not change the binding conformation of the cysteine-rich domain and hence, possibly does not alter its function. The novel anti-proliferative activity of GST-sFRP1 was demonstrated in a dose-dependent manner on two cancer cell lines, viz., HeLa (cervical cancer) and MCF-7 (breast cancer). Also, combination therapy of the protein with chemotherapeutic drugs resulted in enhanced anti-cancer activity. This opens up a new avenue in the application of recombinant sFRP1 for cancer therapeutics. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full text from SpringerLink