title: Use of the Nanofitin Alternative Scaffold as a GFP-Ready Fusion Tag.
abstract: With the continuous diversification of recombinant DNA technologies, the possibilities for new tailor-made protein engineering have extended on an on-going basis. Among these strategies, the use of the green fluorescent protein (GFP) as a fusion domain has been widely adopted for cellular imaging and protein localization. Following the lead of the direct head-to-tail fusion of GFP, we proposed to provide additional features to recombinant proteins by genetic fusion of artificially derived binders. Thus, we reported a GFP-ready fusion tag consisting of a small and robust fusion-friendly anti-GFP Nanofitin binding domain as a proof-of-concept. While limiting steric effects on the carrier, the GFP-ready tag allows the capture of GFP or its blue (BFP), cyan (CFP) and yellow (YFP) alternatives. Here, we described the generation of the GFP-ready tag from the selection of a Nanofitin variant binding to the GFP and its spectral variants with a nanomolar affinity, while displaying a remarkable folding stability, as demonstrated by its full resistance upon thermal sterilization process or the full chemical synthesis of Nanofitins. To illustrate the potential of the Nanofitin-based tag as a fusion partner, we compared the expression level in Escherichia coli and activity profile of recombinant human tumor necrosis factor alpha (TNFα) constructs, fused to a SUMO or GFP-ready tag. Very similar expression levels were found with the two fusion technologies. Both domains of the GFP-ready tagged TNFα were proved fully active in ELISA and interferometry binding assays, allowing the simultaneous capture by an anti-TNFα antibody and binding to the GFP, and its spectral mutants. The GFP-ready tag was also shown inert in a L929 cell based assay, demonstrating the potent TNFα mediated apoptosis induction by the GFP-ready tagged TNFα. Eventually, we proposed the GFP-ready tag as a versatile capture and labeling system in addition to expected applications of anti-GFP Nanofitins (as illustrated with previously described state-of-the-art anti-GFP binders applied to living cells and in vitro applications). Through a single fusion domain, the GFP-ready tagged proteins benefit from subsequent customization within a wide range of fluorescence spectra upon indirect binding of a chosen GFP variant.
authors.affiliation: Affilogic SAS, Nantes, France.
authors.forename: Simon
authors.initials: S
authors.lastname: Huet
authors.affiliation: Affilogic SAS, Nantes, France.
authors.forename: Harmony
authors.initials: H
authors.lastname: Gorre
authors.affiliation: Affilogic SAS, Nantes, France.
authors.forename: Anaëlle
authors.initials: A
authors.lastname: Perrocheau
authors.affiliation: Affilogic SAS, Nantes, France.
authors.forename: Justine
authors.initials: J
authors.lastname: Picot
authors.affiliation: Affilogic SAS, Nantes, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Cinier
mesh_terms: D017209:Apoptosis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D005818:Genetic Engineering / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins