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Features BioSpotlight Playing tag with proteins Non-chromatographic methods for protein purification are attractive in light of the expense and time required for chromatography Perhaps the most promising of these approaches has been the use of protein or peptide stimulus-responsive tags that allow the rapid and reversible precipitation of target proteins from complex solutions After isolation of the fusion protein, the tag can be removed by inducing a coupled self-cleaving intein domain or through treatment with a site-specific protease A number of these tags, however, require heat or high salt treatment to induce their precipitation, which can be potentially deleterious for some target proteins In this month’s issue of BioTechniques, S Banta and colleagues at Columbia University (New York, NY) describe a gentler approach using a synthetic peptide tag they developed that can be reversibly precipitated in response to calcium The new tag came about as a serendipitous discovery during the authors’ research into repeat scaffolds for stimulus-responsive protein engineering applications The calcium-responsive repeat-in-toxin (RTX) domain is found in various bacterial proteins secreted through the type I system and consists of repeats of a nine-amino-acid sequence After Vol 54 | No | 2013 achieve increased solubility, so LaCava Cto and his team tested alternative substitu- NH2 Competitive Displacement of SpA OH NH O NH N O H N O H N HN H N HN O O HN NH2 OH O O S O O B Elution Reagent Solubility 2.73 2.36 2.55 2.22 1.18 RT 0.86 ºC Reagent PEGylOx structure and effectiveness as an elution reagent 180 PEGylOx 25 See20 “Improved native isolation of endogenous protein-A tagged protein 15 complexes” on page 213 10 Written by Patrick Lo, Ph.D., and Nathan Blow, Ph.D.10 20 30 40 50 x lO Gy Gy lO x RPE PE oO x % SpA Retained 50 NH O NH NH N H N N H O HN O O Buffer PEGylOx BioOx 25 00 S 00 O 20 O NH HO 00 O PEGylOx 15 O O 100 at that same N-terminal portion tions 90 peptide, identifying an additional of the 80 polyethylene glycol (PEG) moiety of four70 unit lengths that was most effective 60 in increasing solubility Comparisons PEGylOx IC 50 166 µM 50 between Bio-Ox the newly generated BioOx ICand 50 159 µM 40 peptide PEGylOx showed that PEGylOx 30 effectively release 60%-85% of two could test20protein complexes within 15 minutes 10 while Bio-Ox was unable to release either of these complexes within that time period The authors also demonstrated Concentration of Reagent (μM) that, similar to the original Bio-Ox, PEGylOx could be removed from a sample using a simple spin column with D a 40kDa molecular weight cutoff for downstream assays This newly improved Competitive Displacement of SpA 40 peptide should prove highly advantageous to the 35 large number of researchers taking advantage of SpA-tags in their affinity 30 isolation workflows 10 00 O % SpA Retained O Bi Affinity isolation of protein complexes can be greatly enhanced by speed–the faster the time between isolation and a downstream assay, the better the chance everything is completely recovered in a native state One widely used affinity isolation system is based on the interaction between S aureus Protein-A (SpA) and immunoglobulin G (IgG) Here, a bait protein is tagged with SpA and interacting partners are isolated using either competitive elution or cleavage When it comes to cleavage, the reaction may not be uniform and also tends to be rather slow On the other hand, while competitive elution is more uniform, it also requires several hours of incubation In this issue of BioTechniques, John LaCava and his colleagues from Rockefeller University (New York, NY) describe the design of an improved reagent for the competitive elution of SpA tagged native complexes that works within 15 minutes under very mild conditions Previously, the authors described a modified peptide generated from the Fc fragment of IgG called Bio-Ox that requires a 2-hour or longer incubation for competitive elution They reasoned that by increasing the solubility of this peptide, it might be possible to reduce the elution time The original Bio-Ox peptide was modified at the N terminus See “A designed, phase-changing RTX-based peptide for efficient bioseparations” on page 197 A Concentration (mM) Biochemistry’s 15-minute workout designing a consensus RTX repeat sequence, the authors found that constructs containing multiple repeats of the consensus unit fused to the C-terminus of maltose-binding protein (MBP) caused its precipitation in the presence of calcium Precipitation was most efficient for constructs with 13 or 17 consensus repeats and was easily reversed upon the addition of EGTA Of several cations tested, only calcium could induce precipitation when the 17-repeat tag was fused to various target proteins High yields of all the fusion proteins were obtained, and the functionality of the fused target proteins was also retained To increase the utility of this tag, an enterokinase cleavage site was engineered between the tag and the target protein Cleavage of the isolated fusion protein with enterokinase, followed by calcium-induced precipitation of the tag, left the purified target protein in solution This new calcium-precipitable tag should prove to be a welcome and valuable new method for the rapid and selective purification of recombinant proteins Time of Elution (min) BioTechniques 54:180 (April 2013) doi10.2144/000114004 www.BioTechniques.com 60