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Peptide Linkers (peptide + linker)
Selected AbstractsThe hinge region fragment of immunoglobulin G improves immunogenicity of recombinant gonadotrophin-releasing hormone conjugated to the T-helper epitope in designing peptide vaccinesIMMUNOLOGY, Issue 1pt2 2009Jinshu Xu Summary In our previous study, the hinge fragment (225,232/225,,232,) of human immunoglobulin G1 (IgG1) was used as a space peptide linker for synthesizing the GnRH3,hinge,MVP chimeric peptide, whereby three repeated gonadotrophin-releasing hormone (GnRH) units and a T-cell epitope from measles virus fusion protein (MVP) were amide-bond-linked at the N and C terminus, respectively, to the hinge peptide for producing anti-GnRH antibody responses. To investigate whether or not the hinge region fragment can improve the immunogenicity of GnRH, we further synthesized and purified GnRH3,hinge,MVP, GnRH3,hinge and GnRH3,MVP using recombinant DNA technology. Under high pH conditions, GnRH3,hinge,MVP was capable of forming double-chain structures. Immunization of male mice with the immunogens of GnRH3,hinge,MVP resulted in the generation of high-titre antibodies specific for GnRH. The synthetic GnRH3,hinge and GnRH3,MVP induced a lower titre of anti-GnRH antibody than GnRH3,hinge,MVP. This was followed by a decrease in serum testosterone levels, which resulted in a low level of expression of the relaxin-like factor gene in the testis. Our data suggest that peptide and T-cell epitopes oriented at the N-terminus or C-terminus of hinge peptides simplify the antigenic peptide conjugates and may be considered as potential synthetic immunogens. [source] scFv-based fluorogen activating proteins and variable domain inhibitors as fluorescent biosensor platformsBIOTECHNOLOGY JOURNAL, Issue 9 2009Crystal N. Falco Abstract Single chain antibodies (scFvs) are engineered proteins composed of IgG variable heavy (VH) and variable light (VL) domains tethered together by a flexible peptide linker. We have characterized the individual VH or VL domain activities of several scFvs isolated from a yeast surface-display library for their ability to bind environmentally sensitive fluorogenic dyes causing them to fluoresce. For many of the scFvs, both VH and VL domains are required for dye binding and fluorescence. The analysis of other scFvs, however, revealed that either the VH or the VL domain alone is sufficient to cause the fluorogenic dye activation. Furthermore, the inactive complementary domains in the original scFvs either contribute nothing to, or actually inhibit the activity of these active single domains. We have explored the interactions between active variable domains and inactive complementary domains by extensive variable domain swapping through in vitro gene manipulations to create hybrid scFvs. In this study, we demonstrate that significant alteration of the fluorogenic dye activation by the active VH or VL domains can occur by partnering with different VH or VL complementary domains in the scFv format. Hybrid scFvs can be generated that have fluorogen-activating domains that are completely inhibited by interactions with other domains. Such hybrid scFvs are excellent platforms for the development of several types of genetically encoded, fluorescence-generating biosensors. [source] Nanoarrays: Cooperative Near-Field Surface Plasmon Enhanced Quantum Dot Nanoarrays (Adv. Funct.ADVANCED FUNCTIONAL MATERIALS, Issue 16 2010Mater. Abstract Fluorescence from quantum dots (QDs) sandwiched between colloidal gold nanoparticles and lithographically created metal nanoarrays is studied using engineered peptides as binding agents. For optimized structures, a 15-fold increase is observed in the brightness of the QDs due to plasmon-enhanced fluorescence. This enhanced brightness is achieved by systematically tuning the vertical distance of the QD from the gold nanoparticles using solid-specific peptide linkers and by optimizing the localized surface plasmon resonance by varying the geometric arrangement of the patterned gold nanoarray. The size and pitch of the patterned array affect the observed enhancement, and sandwiching the QDs between the patterned features and colloidal gold nanoparticles yields even larger enhancements due to the increase in local electromagnetic hot spots induced by the increased surface roughness. The use of bifunctional biomolecular linkers to control the formation of hot spots in sandwich structures provides new ways to fabricate hybrid nanomaterials of architecturally induced functionality for biotechnology and photonics. [source] Cooperative Near-Field Surface Plasmon Enhanced Quantum Dot NanoarraysADVANCED FUNCTIONAL MATERIALS, Issue 16 2010Kirsty Leong Abstract Fluorescence from quantum dots (QDs) sandwiched between colloidal gold nanoparticles and lithographically created metal nanoarrays is studied using engineered peptides as binding agents. For optimized structures, a 15-fold increase is observed in the brightness of the QDs due to plasmon-enhanced fluorescence. This enhanced brightness is achieved by systematically tuning the vertical distance of the QD from the gold nanoparticles using solid-specific peptide linkers and by optimizing the localized surface plasmon resonance by varying the geometric arrangement of the patterned gold nanoarray. The size and pitch of the patterned array affect the observed enhancement, and sandwiching the QDs between the patterned features and colloidal gold nanoparticles yields even larger enhancements due to the increase in local electromagnetic hot spots induced by the increased surface roughness. The use of bifunctional biomolecular linkers to control the formation of hot spots in sandwich structures provides new ways to fabricate hybrid nanomaterials of architecturally induced functionality for biotechnology and photonics. [source] Modular, self-assembling peptide linkers for stable and regenerable carbon nanotube biosensor interfaces,JOURNAL OF MOLECULAR RECOGNITION, Issue 4 2006Mark R. Contarino Abstract As part of an effort to develop nanoelectronic sensors for biological targets, we tested the potential to incorporate coiled coils as metallized, self-assembling, site-specific molecular linkers on carbon nanotubes (CNTs). Based on a previously conceived modular anchor-probe approach, a system was designed in which hydrophobic residues (valines and leucines) form the interface between the two helical peptide components. Charged residues (glutamates and arginines) on the borders of the hydrophobic interface increase peptide solubility, and provide stability and specificity for anchor-probe assembly. Two histidine residues oriented on the exposed hydrophilic exterior of each peptide were included as chelating sites for metal ions such as cobalt. Cysteines were incorporated at the peptide termini for oriented, thiol-mediated coupling to surface plasmon resonance (SPR) biosensor surfaces, gold nanoparticles or CNT substrates. The two peptides were produced by solid phase peptide synthesis using Fmoc chemistry: an acidic 42-residue peptide E42C, and its counterpart in the heterodimer, a basic 39-residue peptide R39C. The ability of E42C and R39C to bind cobalt was demonstrated by immobilized metal affinity chromatography and isothermal titration calorimetry. SPR biosensor kinetic analysis of dimer assembly revealed apparent sub-nanomolar affinities in buffers with and without 1,mM CoCl2 using two different reference surfaces. For device-oriented CNT immobilization, R39C was covalently anchored to CNT tips via a C-terminal cysteine residue. Scanning electron microscopy was used to visualize the assembly of probe peptide (E42C) N-terminally labeled with 15,nm gold nanoparticles, when added to the R39C-CNT surface. The results obtained open the way to develop CNT tip-directed recognition surfaces, using recombinant and chemically synthesized chimeras containing binding epitopes fused to the E42C sequence domain. Copyright © 2006 John Wiley & Sons, Ltd. [source] Polymeric Photosensitizer Prodrugs for Photodynamic TherapyPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2007Marino A. Campo ABSTRACT A targeting strategy based on the selective enzyme-mediated activation of polymeric photosensitizer prodrugs (PPP) within pathological tissue has led to the development of agents with the dual ability to detect and treat cancer. Herein, a detailed study of a simple model system for these prodrugs is described. We prepared "first-generation" PPP by directly tethering the photosensitizer (PS) pheophorbide a to poly-(l)-lysine via epsilon amide links and observed that by increasing the number of PS on a polymer chain, energy transfer between PS units improved leading to better quenching efficiency. Fragmentation of the PPP backbone by trypsin digestion gave rise to a pronounced fluorescence increase and to more efficient generation of reactive oxygen species upon light irradiation. In vitro tests using the T-24 bladder carcinoma cell line and ex vivo experiments using mouse intestines illustrated the remarkable and selective ability of these PPP to fluoresce and induce phototoxicity upon enzymatic activation. This work elucidated the basic physicochemical parameters, such as water solubility and quenching/activation behavior, required for the future elaboration of more adaptable "second-generation" PPP, in which the PS is tethered to a proteolytically stable polymer backbone via enzyme-specific peptide linkers. This polymer architecture offers great flexibility to tailor make the PPP to target any pathological tissue known to over-express a specific enzyme. [source] Structure of a fibronectin type III-like module from Clostridium thermocellumACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Markus Alahuhta The 1.6,Å resolution structure of a fibronectin type III-like module from Clostridium thermocellum (PDB code 3mpc) with two molecules in the asymmetric unit is reported. The crystals used for data collection belonged to space group P212121, with unit-cell parameters a = 35.43, b = 45.73, c = 107.72,Å, and the structure was refined to an R factor of 0.166. Structural comparisons found over 800 similar structures in the Protein Data Bank. The broad range of different proteins or protein domains with high structural similarity makes it especially demanding to classify these proteins. Previous studies of fibronectin type III-like modules have indicated that they might function as ligand-binding modules, as a compact form of peptide linkers or spacers between other domains, as cellulose-disrupting modules or as proteins that help large enzyme complexes remain soluble. [source] FACS-Based Selection of Tandem Tetracysteine Peptides with Improved ReAsH Brightness in Live CellsCHEMBIOCHEM, Issue 4 2010Schuyler B. Van Engelenburg Reintroducing ReAsH: Three tandem tetracysteine motifs were optimized for higher-order biarsenical fluorophore binding and enhanced brightness. FACS-based in vivo selections of peptide linkers uncovered new 3×tetracysteine motifs with ,2.5-fold increased brightness in vitro and ,twofold increased brightness in live cells. [source] | |||||||||||||