12 research outputs found
Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay
Site-specific proteolysis of proteins plays an important role in many cellular functions and is often key to the virulence of infectious organisms. Efficient methods for characterization of proteases and their substrates will therefore help us understand these fundamental processes and thereby hopefully point towards new therapeutic strategies. Here, a novel whole-cell in vivo method was used to investigate the substrate preference of the sequence specific tobacco etch virus protease (TEVp). The assay, which utilizes protease-mediated intracellular rescue of genetically encoded short-lived fluorescent substrate reporters to enhance the fluorescence of the entire cell, allowed subtle differences in the processing efficiency of closely related substrate peptides to be detected. Quantitative screening of large combinatorial substrate libraries, through flow cytometry analysis and cell sorting, enabled identification of optimal substrates for TEVp. The peptide, ENLYFQG, identical to the protease's natural substrate peptide, emerged as a strong consensus cleavage sequence, and position P3 (tyrosine, Y) and P1 (glutamine, Q) within the substrate peptide were confirmed as being the most important specificity determinants. In position P1′, glycine (G), serine (S), cysteine (C), alanine (A) and arginine (R) were among the most prevalent residues observed, all known to generate functional TEVp substrates and largely in line with other published studies stating that there is a strong preference for short aliphatic residues in this position. Interestingly, given the complex hydrogen-bonding network that the P6 glutamate (E) is engaged in within the substrate-enzyme complex, an unexpectedly relaxed residue preference was revealed for this position, which has not been reported earlier. Thus, in the light of our results, we believe that our assay, besides enabling protease substrate profiling, also may serve as a highly competitive platform for directed evolution of proteases and their substrates
Scaffold Diversification Enhances Effectiveness of a Superlibrary of Hyperthermophilic Proteins
Screening Libraries To Identify Proteins with Desired Binding Activities Using a Split-GFP Reassembly Assay
Elimination of Redundant and Stop Codons during the Chemical Synthesis of Degenerate Oligonucleotides. Combinatorial Testing on the Chromophore Region of the Red Fluorescent Protein mKate
Retro-inversal of intracellular selected ?-amyloid-interacting peptides: implications for a novel Alzheimer's disease treatment
The aggregation of ?-amyloid (A?) into toxic oligomers is a hallmark of Alzheimer's disease pathology. Here we present a novel approach for the development of peptides capable of preventing amyloid aggregation based upon the previous selection of natural all-l peptides that bind A?1-42. Using an intracellular selection system, successful library members were further screened via competition selection to identify the most effective peptides capable of reducing amyloid levels. To circumvent potential issues arising from stability and protease action for these structures, we have replaced all l residues with d residues and inverted the sequence. These retro-inverso (RI) peptide analogues therefore encompass reversed sequences that maintain the overall topological order of the native peptides. Our results demonstrate that efficacy in blocking and reversing amyloid formation is maintained while introducing desirable properties to the peptides. Thioflavin-T assays, circular dichroism, and oblique angle fluorescence microscopy collectively indicate that RI peptides can reduce amyloid load, while 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays demonstrate modest reductions in cell toxicity. These conclusions are reinforced using Drosophila melanogaster studies to monitor pupal hatching rates and fly locomotor activity in the presence of RI peptides delivered via RI-trans-activating transcriptional activator peptide fusions. We demonstrate that the RI-protein fragment complementation assay approach can be used as a generalized method for deriving A?-interacting peptides. This approach has subsequently led to several peptide candidates being further explored as potential treatments for Alzheimer's disease
A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules
The use of phage display in the study of receptors and their ligands
Phage display technology presents a rapid means by which proteins and peptides that bind specifically to predefined molecular targets can be isolated from extremely complex combinatorial libraries. There are several important ways by which phage display can provide impetus to receptor-based research. Firstly, phage display can be applied, alongside transcriptome and proteome expression profiling techniques, to the identification and characterisation of receptors whose expression is specific to either a cell lineage, a tissue or a disease state. Secondly, specific monoclonal antibodies that enable researchers to identify, localize and quantify receptors can be produced very rapidly (weeks). Thirdly, it should be possible to apply phage display to the matching of orphan ligands and receptors. Finally, phage display can be used to identify proteins and peptides that modulate receptor activity. As well as being useful in the study of receptor function, biologically active proteins and peptides could also be used therapeutically, or as leads for drug design. Hence phage display is ready to play a central role in the study of receptors in the post-genome era. This review outlines the ways in which phage display has been applied to the study of receptor-ligand systems, and discusses how new developments in the technology may be of even greater utility to the field in the next decade