Assessing the bioactivity of the codon optimized sfGFP-IGF1 fusion protein via interaction with IGFBP3 and induction of cell proliferation

Abstract

BackgroundHuman insulin like growth factor-1 (IGF1) is a hormonal peptide associated with growth and development in mammalian. It has become a particularly attractive therapeutic target because of its role in various physiological processes. IGF1 binding to its receptor IGF1R on the surface of the cell triggers a signaling cascade leading to proliferative and anti-apoptotic events. ResultsWe constructed an adapter of codon optimized IGF1 gene with two different linkers; rigid: EAAAK (E) and flexible: GSGSG (G), which was cloned in the plasmid pRSET-sfGFP as a fusion partner to the superfolder form of green fluorescent protein (sfGFP), resulting in the constructs pRSET-sfGFP-IGF1-(E or G). The expressed 6× His tagged sfGFP-IGF1-E protein (36 kDa) was purified from the cytoplasm of E. coli by metal affinity chromatography. Its purity was confirmed by SDS-PAGE blue staining and immunoblotting with anti-His or anti-GFP antibodies. Proper folding of sfGFP-IGF1-E was confirmed by indirect ELISA that revealed the ability of the sfGFP-IGF1-E but not the sfGFP, to bind to the immobilized IGF binding protein 3 (IGFBP3), and the ability of IGFBP3 to bind to the immobilized sfGFP-IGF1-E. Both interactions were able to be detected by anti-GFP and anti-IGFBP3, respectively. XTT test confirmed the bioactivity of sfGFP-IGF1-E after MCF-7 and HepG2 cancer cell lines treatment with increased concentrations of sfGFP-IGF1-E. ConclusionsThe procedure described in this study facilitates the production of effectively sfGFP-IGF1-E fusion protein in sufficiently large amounts, which provides a promising curative compound for many disorders.