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
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Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes
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References
More filters
Journal ArticleDOI
Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides
Journal ArticleDOI
HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis
Georgyi V. Los,Lance P. Encell,Mark McDougall,Danette Hartzell,Natasha Karassina,Chad Zimprich,Monika G. Wood,Randy Learish,Rachel Friedman Ohana,Marjeta Urh,Daniel J. Simpson,Jacqui Méndez,Kris Zimmerman,Paul Otto,Gediminas Vidugiris,Ji Zhu,Aldis Darzins,Dieter Klaubert,Robert F. Bulleit,Keith V. Wood +19 more
TL;DR: The utility of this modular protein tagging system for cellular imaging and protein immobilization is demonstrated by analyzing multiple molecular processes associated with NF-kappaB-mediated cellular physiology, including imaging of subcellular protein translocation and capture of protein--protein and protein--DNA complexes.
Journal ArticleDOI
A general method for the covalent labeling of fusion proteins with small molecules in vivo
TL;DR: A general method for the covalent labeling of fusion proteins in vivo that complements existing methods for noncovalentlabeling of proteins and that may open up new ways of studying proteins in living cells is described.
Journal ArticleDOI
Staphylococcus aureus Sortase, an Enzyme that Anchors Surface Proteins to the Cell Wall
TL;DR: The protein specified by srtA, sortase, may be a useful target for the development of new antimicrobial drugs.
Journal ArticleDOI
An Engineered Protein Tag for Multiprotein Labeling in Living Cells
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).