113231-05-3Relevant articles and documents
Specific and stable fluorescence labeling of histidine-tagged proteins for dissecting multi-protein complex formation
Lata, Suman,Gavutis, Martynas,Tampe, Robert,Piehler, Jacob
, p. 2365 - 2372 (2006)
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.
Specifically and reversibly immobilizing proteins/enzymes to nitriolotriacetic-acid-modified mesoporous silicas through histidine tags for purification or catalysis
Lin, Yu-Chung,Liang, Ming-Ren,Lin, Yu-Chen,Chen, Chao-Tsen
supporting information; experimental part, p. 13059 - 13067 (2012/01/02)
Six nitriolotriacetic-acid-modified ordered mesoporous silicas (NTA-OMPSs) with different pore sizes and surface features for specific and reversible protein immobilization were fabricated and characterized. Specific immobilization of a genetically engineered undecaprenyl pyrophosphate synthase (UPPs) from cell lysate and a chemically modified His-tagged horseradish peroxidase (HRP) in these Ni-NTA-OMPSs through histidine coordination to the nickelated NTA was demonstrated and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Negligible leakage of these enzymes over a wide range of acidic conditions was observed. Moreover, histidine tags with different lengths (His6, His4, His3, and His2) applied to HRP were evaluated to find the minimum length for effective complexation. Enzymatic assessment studies indicated that the pore size of the OMPSs has minimal influence on the enzymatic activity, whereas chemical entities such as unreacted mercapto groups tailored on the interior surfaces of the OMPSs played certain roles in inhibiting the enzymatic activity and stability. On MCF-S-NTA, SBA-S-NTA, and film-S-NTA, which contained unreacted mercaptopropyl groups on the interior surface, immobilized His-tagged HRP showed lower catalytic activity and stability than on MCF-NTA, film-NTA, and SBA-NTA. Selective hydroxylation of optically pure L-tyrosine to (S)-2-amino-3-(3,4-dihydroxyphenyl)propanoic acid (L-DOPA) by the immobilized HRP was also demonstrated. Protein purification and enzyme catalysis: A generic and easily adaptable platform is established for selectively and reversibly immobilizing His-tagged enzymes in ordered mesoporous silicas (OMPSs) through nickelated nitriolotriacetic-acid (NTA)-histidine tag complexation (see figure). The immobilized enzymes exhibit good stability toward heat and pH changes. Negligible leakage of these enzymes over a wide range of acidic conditions was observed. Copyright
Enhanced drug loading in polymerized micellar cargo
Ogier, Julien,Arnauld, Thomas,Carrot, Geraldine,Lhumeau, Antoine,Delbos, Jean-Marie,Boursier, Claire,Loreau, Olivier,Lefoulon, Francois,Doris, Eric
experimental part, p. 3902 - 3907 (2010/09/17)
A new drug carrier system based on self-assembly and polymerization of polydiacetylenic amphiphiles is described. Although classical amphiphiles can help in solubilizing hydrophobic molecules upon self-arrangement into a variety of nanometric structures, a greater effect on drug loading was observed for our polymerized micelles as compared to the non-polymerized analogues. This permitted higher aqueous solubilization of lipophilic drugs with low micelle concentration. 14C labeling of a model drug on one side and of the amphiphile on the other side permitted assessment, after intravenous injection, of biodistribution and excretion profiles of the drug cargo.
