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Use of the Nanofitin Alternative Scaffold as a GFP-Ready Fusion Tag.

Huet S, Gorre H, Perrocheau A, Picot J, Cinier M - PLoS ONE (2015)

Bottom Line: 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.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.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).

View Article: PubMed Central - PubMed

Affiliation: Affilogic SAS, Nantes, France.

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.

No MeSH data available.


Related in: MedlinePlus

Affinity measurement of the Nanofitin D8 against StrepTagII-GFP.Kinetic characterization of StrepTagII-GFP–Anti-GFP Nanofitin D8 interaction, measured by interferometry. Black curves represent experimental data and grey curves represent the statistical fitting of the curves.
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pone.0142304.g004: Affinity measurement of the Nanofitin D8 against StrepTagII-GFP.Kinetic characterization of StrepTagII-GFP–Anti-GFP Nanofitin D8 interaction, measured by interferometry. Black curves represent experimental data and grey curves represent the statistical fitting of the curves.

Mentions: Besides its top rank based on expression yield and its thermal resistance, D8 was initially screened as one of the most affine anti-GFP Nanofitins. This affinity was more precisely determined with a biolayer interferometry assay using an immobilized Nanofitin and a concentration range of GFP (Fig 4). Model fitting to experimental data (R2 = 0.9957) resulted in equilibrium constant of dissociation (KD) equal to 2.52×10−9 M, given the following kinetic constants: kon = 1.86×105 M-1s-1 and koff = 4.67×10−4 s-1.


Use of the Nanofitin Alternative Scaffold as a GFP-Ready Fusion Tag.

Huet S, Gorre H, Perrocheau A, Picot J, Cinier M - PLoS ONE (2015)

Affinity measurement of the Nanofitin D8 against StrepTagII-GFP.Kinetic characterization of StrepTagII-GFP–Anti-GFP Nanofitin D8 interaction, measured by interferometry. Black curves represent experimental data and grey curves represent the statistical fitting of the curves.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4634965&req=5

pone.0142304.g004: Affinity measurement of the Nanofitin D8 against StrepTagII-GFP.Kinetic characterization of StrepTagII-GFP–Anti-GFP Nanofitin D8 interaction, measured by interferometry. Black curves represent experimental data and grey curves represent the statistical fitting of the curves.
Mentions: Besides its top rank based on expression yield and its thermal resistance, D8 was initially screened as one of the most affine anti-GFP Nanofitins. This affinity was more precisely determined with a biolayer interferometry assay using an immobilized Nanofitin and a concentration range of GFP (Fig 4). Model fitting to experimental data (R2 = 0.9957) resulted in equilibrium constant of dissociation (KD) equal to 2.52×10−9 M, given the following kinetic constants: kon = 1.86×105 M-1s-1 and koff = 4.67×10−4 s-1.

Bottom Line: 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.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.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).

View Article: PubMed Central - PubMed

Affiliation: Affilogic SAS, Nantes, France.

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.

No MeSH data available.


Related in: MedlinePlus