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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

Expression of soluble TNFα recombinant proteins in E. coli.(A) SDS-PAGE profiles with 2 μg of purified TNFα fusions, after Coomassie blue staining. Bands indicated with an arrow correspond to the expected recombinant proteins. From left to right: Histag GFP-ready-TNFα (MW: 26.5 kDa), StrepTagII GFP-ready-TNFα (MW: 26.6 kDa), Histag SUMO-TNFα (MW: 30.0 kDa), StrepTagII SUMO-TNFα (MW: 30.1 kDa). (B) Expression yields of Histagged GFP-ready-TNFα and SUMO-TNFα. Quantitation performed by biolayer interferometry with Ni-NTA sensors (n = 4).
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pone.0142304.g005: Expression of soluble TNFα recombinant proteins in E. coli.(A) SDS-PAGE profiles with 2 μg of purified TNFα fusions, after Coomassie blue staining. Bands indicated with an arrow correspond to the expected recombinant proteins. From left to right: Histag GFP-ready-TNFα (MW: 26.5 kDa), StrepTagII GFP-ready-TNFα (MW: 26.6 kDa), Histag SUMO-TNFα (MW: 30.0 kDa), StrepTagII SUMO-TNFα (MW: 30.1 kDa). (B) Expression yields of Histagged GFP-ready-TNFα and SUMO-TNFα. Quantitation performed by biolayer interferometry with Ni-NTA sensors (n = 4).

Mentions: In order to demonstrate the possible use of a Nanofitin as a fusion tag, GFP-ready tagged TNFα (human tumor necrosis factor alpha) was compared to the SUMO (Small Ubiquitin-like Modifier protein) tagged TNFα [38] and recombinant untagged TNFα from a commercial source. We assessed the integrity of the carrier TNFα protein as well as the ability of the GFP-ready tag to maintain its binding properties for the different GFP variants when fused to a partner. GFP-ready-TNFα and SUMO-TNFα were expressed in E. coli in flask and purified to high homogeneity according to the purification tag fused to their N-terminal extremity (Fig 5A). Interestingly, a similar expression yield of more than 60 mg/l of culture was observed regardless of the fusion tag (GFP-ready or SUMO), as determined with interferometry assay (Fig 5B).


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)

Expression of soluble TNFα recombinant proteins in E. coli.(A) SDS-PAGE profiles with 2 μg of purified TNFα fusions, after Coomassie blue staining. Bands indicated with an arrow correspond to the expected recombinant proteins. From left to right: Histag GFP-ready-TNFα (MW: 26.5 kDa), StrepTagII GFP-ready-TNFα (MW: 26.6 kDa), Histag SUMO-TNFα (MW: 30.0 kDa), StrepTagII SUMO-TNFα (MW: 30.1 kDa). (B) Expression yields of Histagged GFP-ready-TNFα and SUMO-TNFα. Quantitation performed by biolayer interferometry with Ni-NTA sensors (n = 4).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142304.g005: Expression of soluble TNFα recombinant proteins in E. coli.(A) SDS-PAGE profiles with 2 μg of purified TNFα fusions, after Coomassie blue staining. Bands indicated with an arrow correspond to the expected recombinant proteins. From left to right: Histag GFP-ready-TNFα (MW: 26.5 kDa), StrepTagII GFP-ready-TNFα (MW: 26.6 kDa), Histag SUMO-TNFα (MW: 30.0 kDa), StrepTagII SUMO-TNFα (MW: 30.1 kDa). (B) Expression yields of Histagged GFP-ready-TNFα and SUMO-TNFα. Quantitation performed by biolayer interferometry with Ni-NTA sensors (n = 4).
Mentions: In order to demonstrate the possible use of a Nanofitin as a fusion tag, GFP-ready tagged TNFα (human tumor necrosis factor alpha) was compared to the SUMO (Small Ubiquitin-like Modifier protein) tagged TNFα [38] and recombinant untagged TNFα from a commercial source. We assessed the integrity of the carrier TNFα protein as well as the ability of the GFP-ready tag to maintain its binding properties for the different GFP variants when fused to a partner. GFP-ready-TNFα and SUMO-TNFα were expressed in E. coli in flask and purified to high homogeneity according to the purification tag fused to their N-terminal extremity (Fig 5A). Interestingly, a similar expression yield of more than 60 mg/l of culture was observed regardless of the fusion tag (GFP-ready or SUMO), as determined with interferometry assay (Fig 5B).

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