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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Dual Site-Specific Chemoenzymatic Antibody Fragment Conjugation Using CRISPR-Based Hybridoma Engineering.
Camille M. Le Gall,Johan M.S. van der Schoot,Iván Ramos-Tomillero,Melek Parlak Khalily,Floris J. van Dalen,Zacharias Wijfjes,Liyan Smeding,Duco van Dalen,Anna Cammarata,Kimberly M. Bonger,Carl G. Figdor,Ferenc A. Scheeren,Martijn Verdoes +12 more
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Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins.
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TL;DR: The protein specified by srtA, sortase, may be a useful target for the development of new antimicrobial drugs.
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Arnaud Gautier,Alexandre Juillerat,Christian Heinis,Ivan R. Corrêa,Maik Kindermann,Florent Beaufils,Kai Johnsson +6 more
TL;DR: The generation of an AGT-based tag is reported, named CLIP-tag, which reacts specifically with O2-benzylcytosine derivatives, which is derived from the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT).