title: Expression, purification, and therapeutic implications of recombinant sFRP1.
abstract: 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.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati-39, Assam, India.
authors.forename: Archita
authors.initials: A
authors.lastname: Ghoshal
authors.forename: Siddhartha Sankar
authors.initials: SS
authors.lastname: Ghosh
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology; D003001:Cloning, Molecular; D004317:Doxorubicin / Q000494:pharmacology; D004357:Drug Synergism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D061986:MCF-7 Cells; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D062105:Molecular Docking Simulation; D017830:Octoxynol / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D012521:Sarcosine / Q000031:analogs & derivatives / Q000737:chemistry; D012995:Solubility; D040681:Structural Homology, Protein; D051153:Wnt Proteins / Q000737:chemistry* / Q000235:genetics; D000094742:Secreted Frizzled-Related Proteins
chemical_list: D000970:Antineoplastic Agents; D002945:Cisplatin; D004317:Doxorubicin; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D017830:Octoxynol; D011993:Recombinant Fusion Proteins; D012521:Sarcosine; D051153:Wnt Proteins; C117636:SFRP1 protein, human; D000094742:Secreted Frizzled-Related Proteins; C092778:WNT7A protein, human; C025231:sarkosyl