Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions
Carla P. Guimaraes,Martin D. Witte,Christopher S. Theile,Gunes Bozkurt,Lenka Kundrat,Annet E M Blom,Hidde L. Ploegh +6 more
TLDR
In this paper, the authors describe the expression and purification conditions for two sortase A enzymes that have different recognition sequences and provide a protocol that allows the functionalization of any given protein at its C terminus, or, for select proteins, at an internal site.Abstract:
Methods for site-specific modification of proteins should be quantitative and versatile with respect to the nature and size of the biological or chemical targets involved. They should require minimal modification of the target, and the underlying reactions should be completed in a reasonable amount of time under physiological conditions. Sortase-mediated transpeptidation reactions meet these criteria and are compatible with other labeling methods. Here we describe the expression and purification conditions for two sortase A enzymes that have different recognition sequences. We also provide a protocol that allows the functionalization of any given protein at its C terminus, or, for select proteins, at an internal site. The target protein is engineered with a sortase-recognition motif (LPXTG) at the place where modification is desired. Upon recognition, sortase cleaves the protein between the threonine and glycine residues, facilitating the attachment of an exogenously added oligoglycine peptide modified with the functional group of choice (e.g., fluorophore, biotin, protein or lipid). Expression and purification of sortase takes ∼3 d, and sortase-mediated reactions take only a few minutes, but reaction times can be extended to increase yields.read more
Citations
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Chemoenzymatic Semisynthesis of Proteins.
Robert E. Thompson,Tom W. Muir +1 more
TL;DR: This review provides a comprehensive survey of the various chemical and enzymatic methods now available for the manufacture of custom proteins containing noncoded elements and discusses the commonalities that exist between these seemingly disparate methods.
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Recent advances in sortase-catalyzed ligation methodology.
TL;DR: An overview of key developments in the practice and implementation of sortase-based strategies, including applications relevant to structural biology are presented.
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Molecular basis of caspase-1 polymerization and its inhibition by a new capping mechanism
Alvin Lu,Yang Li,Yang Li,Florian I. Schmidt,Qian Yin,Qian Yin,Qian Yin,Shuobing Chen,Tian-Min Fu,Tian-Min Fu,Alexander B. Tong,Alexander B. Tong,Hidde L. Ploegh,Youdong Mao,Youdong Mao,Hao Wu,Hao Wu +16 more
TL;DR: The results reveal that INCA caps caspase-1 filaments, thereby exerting potent inhibition with low-nanomolar Ki on casp enzyme-1CARD polymerization in vitro and inflammasome activation in cells.
Journal ArticleDOI
Irreversible Site‐Specific Hydrazinolysis of Proteins by Use of Sortase
TL;DR: Sortase-mediated hydrazinolysis of proteins with hydrazine or its derivatives was developed for the production of recombinant protein hydrazides and provides an alternative method for C-terminal modification of proteinswith functional units as well as for the preparation of C-to-C fusion proteins.
Journal ArticleDOI
Molecular basis of outer kinetochore assembly on CENP-T.
Pim J Huis in 't Veld,Sadasivam Jeganathan,Arsen Petrovic,Priyanka Singh,Juliane John,Veronica Krenn,Florian Weissmann,Tanja Bange,Andrea Musacchio,Andrea Musacchio +9 more
TL;DR: Reconstituted the binding of the MIS12 and NDC80 outer kinetochore subcomplexes to CENP-C and C ENP-T provides a molecular explanation for the stoichiometry of kinetchore components and its cell cycle regulation.
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Arnaud Gautier,Alexandre Juillerat,Christian Heinis,Ivan R. Corrêa,Maik Kindermann,Florent Beaufils,Kai Johnsson +6 more
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