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Patrick Lo, Ph.D. and Nathan Blow, Ph.D.
BioTechniques, Vol. 54, No. 4, April 2013, p. 180
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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 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.

See “A designed, phase-changing RTX-based peptide for efficient bioseparations

Biochemistry's 15-minute workout

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 to achieve increased solubility, so LaCava and his team tested alternative substitutions at that same N-terminal portion of the peptide, identifying an additional polyethylene glycol (PEG) moiety of four unit lengths that was most effective in increasing solubility. Comparisons between Bio-Ox and the newly generated peptide PEGylOx showed that PEGylOx could effectively release 60%-85% of two test protein complexes within 15 minutes while Bio-Ox was unable to release either of these complexes within that time period. The authors also demonstrated that, similar to the original Bio-Ox, PEGylOx could be removed from a sample using a simple spin column with a 40kDa molecular weight cutoff for downstream assays. This newly improved peptide should prove highly advantageous to the large number of researchers taking advantage of SpA-tags in their affinity isolation workflows.

See “Improved native isolation of endogenous protein-A tagged protein complexes