- Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer
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Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly
- Postupalenko, Viktoriia,Desplancq, Dominique,Orlov, Igor,Arntz, Youri,Spehner, Danièle,Mely, Yves,Klaholz, Bruno P.,Schultz, Patrick,Weiss, Etienne,Zuber, Guy
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- Synthesis of a pH-sensitive nitrilotriacetic linker to peptide transduction domains to enable intracellular delivery of histidine imidazole ring-containing macromolecules
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Intracellular delivery of functional macromolecules using peptide transduction domains (PTDs) is an exciting technology with both experimental and therapeutic applications. Recent data indicate that PTD-mediated transduction occurs via fluid-phase macropi
- June, Ronald K.,Gogoi, Khirud,Eguchi, Akiko,Cui, Xian-Shu,Dowdy, Steven F.
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- Oriented insertion of phi29 N-hexahistidine-tagged gp10 connector protein assemblies into C20BAS bolalipid membrane vesicles
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Ni2+:NTA-PEG600-grafted glass surfaces are capable of immobilizing N-his6 gp10 connector protein assemblies from the phi29 DNA packaging motor and mediating their transplantation into bolalipid vesicles whose membrane thickness is co
- Hyun, Seok-Hee,Kim, Hee-Kwon,Kim, Jong-Mok,Thompson, David H.
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- A supramolecular host-guest carrier system for growth factors employing VHH fragments
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A supramolecular strategy is presented for the assembly of growth factors employing His6-tagged single-domain antibodies (VHH). A combination of orthogonal supramolecular interactions of β-cyclodextrin (βCD)-adamantyl (Ad) host-guest
- Cabanas-Dans, Jordi,Rodrigues, Emilie Dooms,Landman, Ellie,Van Weerd, Jasper,Van Blitterswijk, Clemens,Verrips, Theo,Huskens, Jurriaan,Karperien, Marcel,Jonkheijm, Pascal
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- Simple and efficient knockdown of His-tagged proteins by ternary molecules consisting of a His-tag ligand, a ubiquitin ligase ligand, and a cell-penetrating peptide
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We designed and synthesized hybrid molecules for a protein knockdown method based on the recognition of a His-tag fused to a protein of interest (POI). The synthesized target protein degradation inducers contained three functional moieties: a His-tag liga
- Hattori, Takayuki,Okitsu, Koyo,Yamazaki, Norikazu,Ohoka, Nobumichi,Shibata, Norihito,Misawa, Takashi,Kurihara, Masaaki,Demizu, Yosuke,Naito, Mikihiko
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- High-affinity chelator thiols for switchable and oriented immobilization of histidine-tagged proteins: A generic platform for protein chip technologies
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Protein micro/nanoarrays are becoming increasingly important in systematic approaches for the exploration of protein-protein interactions and dynamic protein networks, so there is a high demand for specific, generic, stable, uniform, and locally addressab
- Tinazli, Ali,Tang, Juin,Valiokas, Ramunas,Picuric, Srdjan,Lata, Suman,Piehler, Jacob,Liedberg, Bo,Tampe, Robert
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- Assessing changes in the expression levels of cell surface proteins with a turn-on fluorescent molecular probe
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Tri-nitrilotriacetic acid (NTA)-based fluorescent probes were developed and used to image His-tagged-labelled outer membrane protein C (His-OmpC) in liveEscherichia coli. One of these probes was designed to light up upon binding, which provided the means
- Hatai, Joydev,Prasad, Pragati Kishore,Lahav-Mankovski, Naama,Oppenheimer-Low, Noa,Unger, Tamar,Sirkis, Yael Fridmann,Dadosh, Tali,Motiei, Leila,Margulies, David
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supporting information
p. 1875 - 1878
(2021/03/01)
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- Grid coatings for capture of proteins and other compounds
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Grids comprising a coating modified with one or more capture agents and a deactivating agent are disclosed. Methods of using such grids in connection with suitable microscopy techniques, such as for determining the structure of target compounds including proteins, are also disclosed.
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- Optimized reaction pair of the Cyshis tag and Ni(II)-Nta probe for highly selective chemical labeling of membrane proteins
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Chemical labeling of proteins with synthetic molecular probes offers the possibility to probe the functions of proteins of interest in living cells. However, the methods for covalently labeling targeted proteins using complementary peptide tag-probe pairs are still limited, irrespective of the versatility of such pairs in biological research. Herein, we report the new CysHis tag-Ni(II) probe pair for the specific covalent labeling of proteins. A broad-range evaluation of the reactivity profiles of the probe and the CysHis peptide tag afforded a tag-probe pair with an optimized and high labeling selectivity and reactivity. In particular, the labeling specificity of this pair was notably improved compared to the previously reported one. This pair was successfully utilized for the fluorescence imaging of membrane proteins on the surfaces of living cells, demonstrating its potential utility in biological research.
- Zenmyo, Naoki,Tokumaru, Hiroki,Uchinomiya, Shohei,Fuchida, Hirokazu,Tabata, Shigekazu,Hamachi, Itaru,Shigemoto, Ryuichi,Ojida, Akio
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p. 995 - 1000
(2019/07/18)
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- The Scaffold Design of Trivalent Chelator Heads Dictates Affinity and Stability for Labeling His-tagged Proteins in vitro and in Cells
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Small chemical/biological interaction pairs are at the forefront in tracing protein function and interaction at high signal-to-background ratios in cellular pathways. However, the optimal design of scaffold, linker, and chelator head still deserve systematic investigation to achieve the highest affinity and kinetic stability for in vitro and especially cellular applications. We report on a library of N-nitrilotriacetic acid (NTA)-based multivalent chelator heads (MCHs) built on linear, cyclic, and dendritic scaffolds and compare these with regard to their binding affinity and stability for the labeling of cellular His-tagged proteins. Furthermore, we describe a new approach for tracing cellular target proteins at picomolar probe concentrations in cells. Finally, we outline fundamental differences between the MCH scaffolds and define a cyclic trisNTA chelator that displays the highest affinity and kinetic stability of all reported reversible, low-molecular-weight interaction pairs.
- Gatterdam, Karl,Joest, Eike F.,Gatterdam, Volker,Tampé, Robert
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supporting information
p. 12395 - 12399
(2018/09/18)
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- MOLECULAR SENSOR AND METHODS OF USE THEREOF
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The present invention is directed to fluorescent molecular sensor based on thiazole orange as represented below for protein detection. Interaction of the protein target with the molecular sensors of this invention results in a significant increase in the fluorescence emission. The generation of light output signal enables one to detect protein biomarkers associated with different diseases or detecting the protein of interest also in living cells.
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Paragraph 00118
(2014/07/21)
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- Synthesis of nickel-chelating fluorinated lipids for protein monolayer crystallizations
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Nickel-chelating lipids have been synthesized for use as functionalized templates for 2-D crystallization of membrane proteins. These monolayer-forming lipids have been designed with three distinct components: (i) a branched hydrocarbon tail to confer flu
- Hussein, Waleed M.,Ross, Benjamin P.,Landsberg, Michael J.,Lévy, Daniel,Hankamer, Ben,McGeary, Ross P.
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supporting information; experimental part
p. 1473 - 1479
(2009/07/11)
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- Synthesis and photochemical properties of a light-activated fluorophore to label His-tagged proteins
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Rapid and efficient light-induced fluorescence enhancement is demonstrated on a DMNPB-"caged" coumarin derivative carrying a His-tag recognition motif. The Royal Society of Chemistry.
- Orange, Clelia,Specht, Alexandre,Puliti, David,Sakr, Elias,Furuta, Toshiaki,Winsor, Barbara,Goeldner, Maurice
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p. 1217 - 1219
(2008/12/21)
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- MULTIVALENT CHELATORS FOR MODIFYING AND ORGANIZING OF TARGET MOLECULES
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New compounds of the general formula: Xm-G-Cln are described as well as methods for their production. These constitute multivalent chelator-compounds with an affinity-tag binding to metal-chelator-complexes which can selectively modify and/or immobilize target molecules by a multitude of probes or functional units.
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Page/Page column 26
(2008/06/13)
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- Specific and stable fluorescence labeling of histidine-tagged proteins for dissecting multi-protein complex formation
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Labeling of proteins with fluorescent dyes offers powerful means for monitoring protein interactions in vitro and in live cells. Only a few techniques for noncovalent fluorescence labeling with well-defined localization of the attached dye are currently available. Here, we present an efficient method for site-specific and stable noncovalent fluorescence labeling of histidine-tagged proteins. Different fluorophores were conjugated to a chemical recognition unit bearing three NTA moieties (tris-NTA). In contrast to the transient binding of conventional mono-NTA, the multivalent interaction of tris-NTA conjugated fluorophores with oligohistidine-tagged proteins resulted in complex lifetimes of more than an hour. The high selectivity of tris-NTA toward cumulated histidines enabled selective labeling of proteins in cell lysates and on the surface of live cells. Fluorescence labeling by tris-NTA conjugates was applied for the analysis of a ternary protein complex in solution and on surfaces. Formation of the complex and its stoichiometry was studied by analytical size exclusion chromatography and fluorescence quenching. The individual interactions were dissected on solid supports by using simultaneous mass-sensitive and multicolor fluorescence detection. Using these techniques, formation of a 1:1:1 stoichiometry by independent interactions of the receptor subunits with the ligand was shown. The incorporation of transition metal ions into the labeled proteins upon labeling with tris-NTA fluorophore conjugates provided an additional sensitive spectroscopic reporter for detecting and monitoring protein-protein interactions in real time. A broad application of these fluorescence conjugates for protein interaction analysis can be envisaged.
- Lata, Suman,Gavutis, Martynas,Tampe, Robert,Piehler, Jacob
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p. 2365 - 2372
(2007/10/03)
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- High-affinity adaptors for switchable recognition of histidine-tagged proteins
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We aspired to create chemical recognition units, which bind oligohistidine tags with high affinity and stability, as tools for selectively attaching spectroscopic probes and other functional elements to recombinant proteins. Several supramolecular entities containing 2-4 nitrilotriacetic acid (NTA) moieties were synthesized, which additionally contained an amino group, to which fluorescein was coupled as a sensitive reporter probe. These multivalent chelator heads (MCH) (termed bis-, tris-, and tetrakis-NTA) were characterized with respect to their interaction with hexahistidine (H6)- and decahistidine (H10)-tagged targets. Substantially increased binding stability with increasing number of NTA moieties was observed by analytical size exclusion chromatography. The binding enthalpies as determined by isothermal titration calorimetry increased nearly additively with the number of possible coordinative bonds between chelator heads and tags. Yet, a substantial excess of histidines in the oligohistidine tag was required for obtaining fully additive binding enthalpies. Dissociation kinetics of MCH/oligohistidine complexes measured by fluorescence dequenching showed an increase in stability by 4 orders of magnitude compared to that of mono-NTA, and subnanomolar affinity was reached for tris-NTA. The gain in free energy with increasing multivalency was accompanied by an increasing loss of entropy, which was ascribed to the high flexibility of the binding partners. Numerous applications of these MCHs for noncovalent, high affinity, yet reversible tethering of spectroscopic probes and other functional elements to the recombinant proteins can be envisioned.
- Lata, Suman,Reichel, Annett,Brock, Roland,Tampe, Robert,Piehler, Jacob
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p. 10205 - 10215
(2007/10/03)
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- Molecular recognition of histidine-tagged molecules by metal-chelating lipids monitored by fluorescence energy transfer and correlation spectroscopy
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Complex binding of proteins by metal-chelating lipids via surface- exposed or protein-engineered histidines provides an universal and powerful concept for the orientation and two-dimensional crystallization of proteins at self-organized interfaces. To dem
- Dorn, Ingmar T.,Neumaier, Klaus R.,Tampé, Robert
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p. 2753 - 2763
(2007/10/03)
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