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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Journal ArticleDOI
Enzyme‐mediated assembly of chimeric membrane proteins at phospholipid bilayers
D. Scott,D. Scott,Jennifer Tullman,Jennifer Tullman,Zvi Kelman,Zvi Kelman,J. Marino,J. Marino,Vitalii Silin,Vitalii Silin +9 more
TL;DR: A generalized strategy for the assembly of chimeric IMPs within a phospholipid bilayer surface based on a two‐step process that first imbeds the insoluble domain of the IMP into a phosphoipid layer and then ligates the soluble part of theIMP to the imbedded portion under mild biochemical conditions using a mutant sortase A enzyme.
Posted ContentDOI
A second specificity-determining loop in Class A sortases: Biochemical characterization of natural sequence variation in chimeric SrtA enzymes
Isabel M. Piper,Sarah A. Struyvenberg,Jordan D. Valgardson,Johnson Da,Melody Gao,Johnston K,Justin E. Svendsen,Hanna M. Kodama,Kelli L. Hvorecny,John M. Antos,Jeanine F. Amacher +10 more
TL;DR: In this paper, the authors used principal component analysis (PCA) to identify a structurally conserved loop with a high degree of variability in all classes of sortase enzymes.
Patent
Novel soluble sortase a
TL;DR: In this paper, a polypeptide comprising the amino acid sequence of SEQ ID NO: 38 was reported as sole Listeria monocytogenes derived polyprotein.
Exploration of Sortase A-Inhibitor Binding Mechanisms Through Isothermal Titration Calorimetry
TL;DR: The thermodynamic characteristics and binding mechanism of Sortase A and inhibitors using isothermal titration calorimetry support a two-step, sequential binding mechanism involving induced fit and conformational change.
Patent
In vitro production of red blood cells with proteins comprising sortase recognition motifs
TL;DR: In this paper, surface modified enucleated red blood cells, e.g., conjugated with an agent of interest such as a peptide, a detectable label, or a chemotherapeutic agent, and uses thereof in delivering the agent to a subject are described.
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).