Many proteins canâ??t be studied using solution NMR methods because they have limited solubility. To overcome this problem, recalcitrant proteins can be fused to a more soluble protein that functions as a solubility tag. However, signals arising from the solubility tag hinder data analysis because they increase spectral complexity. We report a new method to rapidly and efficiently add a non-isotopically labeled Small Ubiquitin-like Modifier protein (SUMO) solubility tag to an isotopically labeled protein. The method makes use of a newly developed SUMO-Sortase tagging reagent in which SUMO and the Sortase A (SrtA) enzyme are present within the same polypeptide. The SUMO-Sortase reagent rapidly attaches SUMO to any protein that contains the sequence LPXTG at its C-terminus. It modifies proteins at least 15-times faster than previously described approaches, and does not require active dialysis or centrifugation during the reaction to increase product yields. In addition, silently tagged proteins are readily purified using the well-established SUMO expression and purification system. The utility of the SUMO-Sortase tagging reagent is demonstrated using PhoP and green fluorescent proteins, which are ~90Â*% modified with SUMO at room temperature within four hours. SrtA is widely used as a tool to construct bioconjugates. Significant rate enhancements in these procedures may also be achieved by fusing the sortase enzyme to its nucleophile substrate.
Efficient segmental isotope labeling of multi-domain proteins using Sortase A
Efficient segmental isotope labeling of multi-domain proteins using Sortase A
Abstract
NMR studies of multi-domain protein complexes provide unique insight into their molecular interactions and dynamics in solution. For large proteins domain-selective isotope labeling is desired to reduce signal overlap, but available methods require extensive optimization and often give poor ligation yields. We present an optimized strategy for segmental labeling of multi-domain proteins using the S. aureus transpeptidase Sortase A. Critical improvements compared to...
Engineered solubility tag for solution NMR of proteins
Engineered solubility tag for solution NMR of proteins
Abstract
The low solubility of many proteins hinders large scale expression and purification as well as biophysical measurements. Here, we devised a general strategy to solubilize a protein by conjugating it at a solvent-exposed position to a 6 kDa protein that was re-engineered to be highly soluble. We applied this method to the CARD domain of Apoptosis-associated speck-like protein containing a CARD (ASC), which represents one member of a class of proteins that are notoriously prone to aggregation. Attachment of the tag to a...
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09-20-2013 02:39 PM
Rapid Injection NMR Reveals ?3 ‘?-Allyl’ CuIII Intermediates in Addition Reactions of Organocuprate Reagents
Rapid Injection NMR Reveals ?3 ‘?-Allyl’ CuIII Intermediates in Addition Reactions of Organocuprate Reagents
Steven H. Bertz, Richard A. Hardin, Michael D. Murphy, Craig A. Ogle, Joshua D. Richter and Andy A. Thomas
http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/0/jacsat.ahead-of-print/ja3022363/aop/images/medium/ja-2012-022363_0012.gif
Journal of the American Chemical Society
DOI: 10.1021/ja3022363
http://feeds.feedburner.com/~ff/acs/jacsat?d=yIl2AUoC8zA
http://feeds.feedburner.com/~r/acs/jacsat/~4/FjoEID-4z0g
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05-25-2012 07:14 PM
Overcoming the solubility limit with solubility-enhancement tags: successful applications in biomolecular NMR studies
Overcoming the solubility limit with solubility-enhancement tags: successful applications in biomolecular NMR studies
Abstract Although the rapid progress of NMR technology has significantly expanded the range of NMR-trackable systems, preparation of NMR-suitable samples that are highly soluble and stable remains a bottleneck for studies of many biological systems. The application of solubility-enhancement tags (SETs) has been highly effective in overcoming solubility and sample stability issues and has enabled structural studies of important biological systems previously deemed...
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01-09-2011 12:46 PM
Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy
Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy
Abstract NMR spectroscopy has distinct advantages for providing insight into protein structures, but faces significant resolution challenges as protein size increases. To alleviate such resonance overlap issues, the ability to produce segmentally labeled proteins is beneficial. Here we show that the S. aureus transpeptidase sortase A can be used to catalyze the ligation of two separately expressed domains of the same protein, MecA (B. subtilis). The yield of purified, segmentally...
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12-31-2010 08:38 PM
Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy.
Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy.
Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy.
J Biomol NMR. 2010 Dec 29;
Authors: Refaei MA, Combs A, Kojetin DJ, Cavanagh J, Caperelli C, Rance M, Sapitro J, Tsang P
NMR spectroscopy has distinct advantages for providing insight into protein structures, but faces significant resolution challenges as protein size increases. To alleviate such resonance overlap issues, the ability to...
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12-29-2010 04:04 PM
[NMR paper] Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically l
Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies.
Related Articles Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies.
Nat Biotechnol. 2005 Jun;23(6):736-40
Authors: Züger S, Iwai H
Segmental isotopic labeling of proteins using protein ligation is a recently established in vitro method for incorporating isotopes into one domain or region of a protein to reduce the complexity of NMR spectra, thereby facilitating...