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His-tagged Proteins (His-tagge + protein)
Selected AbstractsFunctional Hydrogel Surfaces: Binding Kinesin-Based Molecular Motor Proteins to Selected Patterned Sites,ADVANCED FUNCTIONAL MATERIALS, Issue 8 2005T. Yu Abstract Hydrogel microstructures with micrometer-scale topography and controllable functionality have great potential for numerous nanobiotechnology applications including, for example, three-dimensional structures that exhibit controlled interactions with proteins and cells. Taking advantage of the strong affinity of histidine (His) residues for metal-ion,nitrilotriacetic acid (NTA) complexes, we have chemically modified hydrogels to enable protein immobilization with retention of activity by incorporating 2-methacrylamidobutyl nitrilotriacetic acid, an NTA-containing monomer that can be copolymerized with a series of monomers to form NTA-containing hydrogels. By varying the NTA-monomer composition in the hydrogels, it is possible to control the amount of protein bound to the hydrogel surface. The retention of biological activity was demonstrated by microtubule gliding assays. Normally, hydrogels are resistant to protein binding, but we have selected these materials because of their porous nature. Bringing together hydrogel functionalization and soft-lithography patterning techniques, it was possible to create a hybrid hydrogel superstructure that possesses binding specificity to His-tagged protein in selected sites. This type of surface and microstructure is not only advantageous for motor protein integration, but it can also be generally applied to the formation of His-tagged molecules for sensors and biochip applications. [source] Activation of enteropathogenic Escherichia coli (EPEC) LEE2 and LEE3 operons by LerMOLECULAR MICROBIOLOGY, Issue 4 2000Vanessa Sperandio Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing lesions (AE) on epithelial cells. The genes involved in the formation of the AE lesions are contained within a pathogenicity island named the locus of enterocyte effacement (LEE). The LEE comprises 41 open reading frames organized in five major operons: LEE1, LEE2, LEE3, LEE4 and tir. The first gene of the LEE1 operon encodes a transcription activator of the other LEE operons that is called the LEE-encoded regulator (Ler). The LEE2 and LEE3 operons are divergently transcribed with overlapping ,10 promoter regions, and gene fusion studies have shown that they are both activated by Ler. Deletion analysis, using lacZ reporter fusions, of the LEE2 and LEE3 promoters demonstrated that deletions extending closer to the LEE2 transcription start site than ,247 bp lead to loss of activation by Ler, whereas only 70 bp upstream of the LEE3 transcription start site is required for Ler-mediated activation. We have purified Ler as a His-tagged protein and used it to perform DNA-binding assays with LEE2 and LEE3. We observed that Ler bound to a DNA fragment containing the ,300 to +1 region of LEE2; however, it failed to bind to a DNA fragment containing the ,300 to +1 region of LEE3, suggesting that Ler activates both operons by only binding to the regulatory region upstream of LEE2. The Ler-activatable LEE3::lacZ fusions extended to what would be ,246 bp of the LEE2 operon. A lacZ fusion from the ,300 to +1 region of LEE3 failed to be activated by Ler, consistent with our hypothesis that Ler activates the expression of LEE2 and LEE3 by binding to a region located downstream of the LEE3 transcription start site. DNase I footprinting revealed that Ler protected a region of 121 bp upstream of LEE2. Purified Ler mutated in the coiled-coil domain was unable to activate transcription and to bind to the LEE2 regulatory region. These data indicate that Ler may bind as a multimer to LEE2 and activate both divergent operons by a novel mechanism potentially involving changes in the DNA structure. [source] Potential for Using Histidine Tags in Purification of Proteins at Large ScaleCHEMICAL ENGINEERING & TECHNOLOGY (CET), Issue 11 2005V. Gaberc-Porekar Abstract Attachment of oligo-histidine tag (His-tag) to the protein N- or C-terminus is a good example of early and successful protein engineering to design a unique and generalized purification scheme for virtually any protein. Thus relatively strong and specific binding of His-tagged protein is achieved on an Immobilized Metal-Ion Affinity Chromatography (IMAC) matrix. Most popular hexa-histidine tag and recently also deca-histidine tag are used in combination with three chelating molecules: iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), and carboxymethyl aspartic acid (CM-Asp), covalently attached to the chromatographic matrix. The following combinations with divalent metal ions are preferentially used: (Cu, Zn, Ni, Co)-IDA, Ni-NTA, and Co-CM-Asp. At large scale, regarding cost and product purity, a decisive step is precise and efficient cleavage of His-tag by the cleavage enzyme. Two-step IMAC followed by a polishing step appears to be a minimum but still realistic as an approach to generic technology also for more demanding products. Possible drawbacks in using His-tags and IMAC, such as leaching of metal ions, inefficient cleavage, and batch-to-batch reproducibility must be carefully evaluated before transferred to large scale. Although a great majority of reports refer to small laboratory scale isolations for research purposes it appears there is much higher potential for more extensive use of His-tags and IMAC at large scale than currently documented. [source] Insertion of light-harvesting chlorophyll a/b protein into the thylakoidFEBS JOURNAL, Issue 4 2000Topographical studies The major light-harvesting chlorophyll a/b -binding protein (Lhcb1,2) of photosystem II is inserted into the thylakoid via the signal recognition particle dependent pathway. However, the mechanism by which the protein enters the membrane is at this time unknown. In order to define some topographical restrictions for this process, we constructed several recombinant derivatives of Lhcb1 carrying hexahistidine tags at either protein terminus or in the stromal loop domain. Additionally, green fluorescent protein (GFP) was fused to either terminus. None of the modifications significantly impair the pigment-binding properties of the protein in the in vitro reconstitution of LHCII. With the exception of the C-terminal GFP fusion, all mutants stably insert into isolated thylakoids in the absence of Ni2+ ions. The addition of low concentrations of Ni2+ ions abolishes the thylakoid insertion of C-terminally His-tagged mutants whereas the other His-tagged proteins fail to insert only at higher Ni2+ concentrations. The C-terminus of Lhcb1 must cross the membrane during protein insertion whereas the other sites of Lhcb1 modification are positioned on the stromal side of LHCII. We conclude that a Ni2+ -complexed His tag and fusion to GFP inhibit translocation of the protein C-terminus across the thylakoid. Our observations indicate that the N-terminal and stromal domain of Lhcb1 need not traverse the thylakoid during protein insertion and are consistent with a loop mechanism in which only the C-terminus and the lumenal loop of Lhcb1 are translocated across the thylakoid. [source] Enzyme-Mediated Deposition of a TiO2 Coating onto Biofunctionalized WS2 Chalcogenide Nanotubes,ADVANCED FUNCTIONAL MATERIALS, Issue 2 2009Muhammad Nawaz Tahir Abstract A chemically specific and facile method for the biofunctionalization of WS2 nanotubes (NT-WS2) is reported. The covalent modification strategy is based on the affinity of the nitrilotriacetic acid (NTA) side chain, which serves as a ligand for the surface binding to NT-WS2 and simultaneously as an anchor group for the binding of His-tagged proteins to the polymer backbone. The polymer functionalized WS2 nanotubes can be solubilized either in water or organic solvents; they are stable for at least one week. The probes were characterized by FT-IR and UV-vis spectroscopy. The immobilization of silicatein, a hydrolytic protein encountered in marine sponges, was visualized by scanning force microscopy (SFM) and confocal laser scanning microscopy (CLSM). The formation of the biotitania coating mediated by the immobilized silicatein onto the surface was characterized by scanning electron microscopy (SEM), and transmission electron microscopy (TEM). [source] Two-dimensional crystals of carboxysome shell proteins recapitulate the hexagonal packing of three-dimensional crystalsPROTEIN SCIENCE, Issue 12 2009Kelly A. Dryden Abstract Bacterial microcompartments (BMCs) are large intracellular bodies that serve as simple organelles in many bacteria. They are proteinaceous structures composed of key enzymes encapsulated by a polyhedral protein shell. In previous studies, the organization of these large shells has been inferred from the conserved packing of the component shell proteins in two-dimensional (2D) layers within the context of three-dimensional (3D) crystals. Here, we show that well-ordered, 2D crystals of carboxysome shell proteins assemble spontaneously when His-tagged proteins bind to a monolayer of nickelated lipid molecules at an air,water interface. The molecular packing within the 2D crystals recapitulates the layered hexagonal sheets observed in 3D crystals. The results reinforce current models for the molecular design of BMC shells. [source] Novel Probes Showing Specific Fluorescence Enhancement on Binding to a Hexahistidine TagCHEMISTRY - A EUROPEAN JOURNAL, Issue 26 2008Mie Kamoto Abstract The introduction of hexahistidine (His tag) is widely used as a tool for affinity purification of recombinant proteins, since the His tag binds selectively to nickel,nitrilotriacetic acid (Ni2+,NTA) complex. To develop efficient "turn-on" fluorescent probes for His-tagged proteins, we adopted a fluorophore displacement strategy, that is, we designed probes in which a hydroxycoumarin fluorophore is joined via a linker to a metal,NTA moiety, with which it forms a weak intramolecular complex, thereby quenching the fluorescence. In the presence of a His tag, with which the metal,NTA moiety binds strongly, the fluorophore is displaced, which results in a dramatic enhancement of fluorescence. We synthesized a series of hydroxycoumarins that were modified by various linkers with NTA (NTAC ligands), and investigated the chemical and photophysical properties of the free ligands and their metal complexes. From the viewpoint of fluorescence quenching, Ni2+ and Co2+ were the best metals. Fluorescence spectroscopy revealed a 1:1 binding stoichiometry for the Ni2+ and Co2+ complexes of NTACs in pH,7.4 aqueous buffer. As anticipated, these complexes showed weak intrinsic fluorescence, but addition of a His-tagged peptide (H-(His)6 -Tyr-NH2; Tyr was included to allow convenient concentration measurement) in pH,7.4 aqueous buffer resulted in up to a 22-fold increase in the fluorescence quantum yield. We found that the Co2+ complexes showed superior properties. No fluorescence enhancement was seen in the presence of angiotensin,I, which contains two nonadjacent histidine residues; this suggests that the probes are selective for the polyhistidine peptide. [source] | ||||||||||