Nouvelles stratégies d'ancrage de protéïnes sur une surface de phosphonate de zirconium

Miniaturisation, automatisation et rapidité d'analyse font partie des challenges actuels de la génomique et plus récemment de la protéomique. Le développement des nanotechnologies dans ces domaines, notamment avec l'avènement des puces biologiques, tente de répondre à cette problématique. Dans ce contexte, nous avons développé deux stratégies pour permettre l'immobilisation dense et orientée de protéines sur un film de phosphonate de zirconium. La première stratégie implique la synthèse de deux adaptateurs bifonctionnels. Pour cela, une ou deux unités NTA ont été couplées à un motif bisphosphonate pour leur immobilisation stable sur une surface de phosphonate de zirconium. Au cours de ce travail de thèse nous avons pu montrer que ces adaptateurs Mono-ou Bis-NTA permettaient de conférer au support de phosphonate de zirconium une nouvelle spécificité pour l'ancrage poly(his)6. La seconde stratégie fait intervenir la conception d'une étiquette comportant un nanocluster de quatre sérines phosphorylables par la caséine kinase II (CKII). Insérée sur la position C-terminale d'une affitine, cette étiquette s'est avérée être complètement et spécifiquement phosphorylée par la CKII in vitro. Noua avons démontré l'efficacité de cette stratégie originale basée sur l'ingénierie d'un nanocluster de phosphate pour l'immobilisation sélective des protéines sur un support de phosphonate de zirconium.Dans les deux cas, l'immobilisation dense et orientée des protéines sur un support à base de phosphonate de zirconium a été démontrée. De plus, cette nouvelle technologie permet la préparation de puces à protéines de haute qualité comparée aux supports commerciaux.Miniaturization, automation and high troughput of analysis belong to the current challenges of the genomics and more recently of the proteomics. Progress of nanotechnologies in these fields, in particular with the advent of the biological chips, aim to solve this problem. In this context, we developped two strategies for dense and orientated protein immobilization onto a zirconium phosphonate coated glasss slide:the first one involve synthesis of two bifunctional adaptors. One or two NTA units were conjugated to a multivalent phosphonic acid dipode which interacts strongly with the zirconated monolayer. Zirconated surface fonctionnalized with these linkers have been demonstrated to provide a new selectivity for histagged protein anchoring.The second one involve engineering of a multiphosphorylable tag by Casein Kinase II (CKII) consisting in a nanocluster of four serines. Merged to the C terminal end of an affitin, this tag was demonstrated to be totally and specifically phosphorylated in vitro by CKII. This original phosphate nanocluster based strategy was demonstrated to be effective for selective protein immobilization onto the zirconated support. These both strategies provided uniform orientation of proteins on the chip surface and a higher density of coupling compared to commercial supports. This new technology provides a very high selectivity of anchoring and exhibits high signal to noise ratio compared to other functionalized supports.NANTES-BU Sciences (441092104) / SudocSudocFranceF

Engineering of a phosphorylatable tag for specific protein binding on zirconium phosphonate based microarrays

A phosphorylatable tag was designed and fused at the C-terminal end of proteins, which allowed efficient and oriented immobilization of capture proteins on glass substrates coated with a zirconium phosphonate monolayer. The concept is demonstrated using Nanofitin directed against lysozyme. This peptide tag (DSDSSSEDE) contains four serines in an acidic environment, which favored its in vitro phosphorylation by casein kinase II. The resulting phosphate cluster at the C-terminal end of the protein provided a specific, irreversible, and multipoint attachment to the zirconium surface. In a microarray format, the high surface coverage led to high fluorescence signal after incubation with Alexa Fluor 647 labeled lysozyme. The detection sensitivity of the microarray for the labeled target was below 50 pM, owing to the exceptionally low background staining, which resulted in high fluorescence similar to signal to noise ratios. The performance of this new anchoring strategy using a zirconium phosphonate modified surface compares favorably with that of other types of microarray substrates, such as nitrocellulose-based or epoxide slides, which bind proteins in a nonoriented way

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

<div><p>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 <i>Escherichia coli</i> 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 <i>in vitro</i> 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.</p></div

Discovery of APL-1030, a Novel, High-Affinity Nanofitin Inhibitor of C3-Mediated Complement Activation

Uncontrolled complement activation contributes to multiple immune pathologies. Although synthetic compstatin derivatives targeting C3 and C3b are robust inhibitors of complement activation, their physicochemical and molecular properties may limit access to specific organs, development of bifunctional moieties, and therapeutic applications requiring transgenic expression. Complement-targeting therapeutics containing only natural amino acids could enable multifunctional pharmacology, gene therapies, and targeted delivery for underserved diseases. A Nanofitin library of hyperthermophilic protein scaffolds was screened using ribosome display for C3/C3b-targeting clones mimicking compstatin pharmacology. APL-1030, a recombinant 64-residue Nanofitin, emerged as the lead candidate. APL-1030 is thermostable, binds C3 (KD, 1.59 nM) and C3b (KD, 1.11 nM), and inhibits complement activation via classical (IC50 = 110.8 nM) and alternative (IC50 = 291.3 nM) pathways in Wieslab assays. Pharmacologic activity (determined by alternative pathway inhibition) was limited to primate species of tested sera. C3b-binding sites of APL-1030 and compstatin were shown to overlap by X-ray crystallography of C3b-bound APL-1030. APL-1030 is a novel, high-affinity inhibitor of primate C3-mediated complement activation developed from natural amino acids on the hyperthermophilic Nanofitin platform. Its properties may support novel drug candidates, enabling bifunctional moieties, gene therapy, and tissue-targeted C3 pharmacologics for diseases with high unmet need

Kinetic constants of Nanofitin D8 directed toward GFP variants.

<p>Kinetic constants of interaction between Nanofitin D8 and GFP variants, determined by interferometry. k_on, the association rate constant, in M^-1s^-1. k_off, the dissociation rate constant in s^-1. K_D, the equilibrium binding constant, in M, computed as k_off/k_on. R², the coefficient of determination of the fitted model.</p><p>Kinetic constants of Nanofitin D8 directed toward GFP variants.</p

Structure of the Nanofitin OB-fold scaffold and randomized libraries.

<p>(A) Cartoon representation of wild-type Sac7d (Protein Data Bank code 1AZP). (B) Secondary structure plot of wild-type Sac7d. Helices are labeled H1, H2 and H3 and strands by their sheets A and B, from N-terminal to C-terminal extremities. Beta-turn motifs are indicated with β. Residues randomized in the library involved in the generation of anti-GFP Nanofitins are shown in (A) yellow sticks and (B) yellow frames. Residues of the first loop randomized in early library are shown in (A) purple sticks and (B) purple frames.</p

Binding of TNFα fusions to anti-TNFα antibody and GFP variants.

<p>Binding of GFP variants to TNFα fusions, measured by ELISA or interferometry with immobilized anti-TNFα antibody, Infliximab. (A) ELISA with no TNFα fusion (white bars), SUMO-TNFα fusion (grey bars) or GFP-ready-TNFα fusion (black bars) captured in plate wells (n = 3). (B) Interferometry kinetic binding profile with loading of Infliximab (step 1), GFP-ready-TNFα fusion (step 2) and GFP (step 3). Besides the sample with all bound partners (plain line), controls without GFP, GFP-ready-TNFα or Infliximab were also measured (dashed line, dotted line and grey line, respectively).</p

Cell growth inhibition of TNFα fusions.

<p>TNFα activity was assessed by measuring cell viability (n = 3) using the XTT assay on L929 sensitized cells with actinomycin-D and concentration range of TNFα fusions (untagged soluble TNFα, SUMO-TNFα or GFP-ready-TNFα from left to right). Individual IC₅₀ values were determined from curve fitting.</p