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
More filters
Journal ArticleDOI
Copper-Mediated Living Radical Polymerization (Atom Transfer Radical Polymerization and Copper(0) Mediated Polymerization): From Fundamentals to Bioapplications
Cyrille Boyer,Nathaniel Corrigan,Kenward Jung,Diep Nguyen,Thuy-Khanh Nguyen,Nik Nik M. Adnan,Susan Oliver,Sivaprakash Shanmugam,Jonathan Yeow +8 more
TL;DR: Radical Polymerization and Copper(0) Mediated polymerization): From Fundamentals to Bioapplications
Journal ArticleDOI
Cryo-EM structure of the activated NAIP2-NLRC4 inflammasome reveals nucleated polymerization.
Liman Zhang,Shuobing Chen,Jianbin Ruan,Jianbin Ruan,Jiayi Wu,Jiayi Wu,Alexander B. Tong,Alexander B. Tong,Qian Yin,Qian Yin,Yang Li,Yang Li,Liron David,Liron David,Alvin Lu,Alvin Lu,Wei Li Wang,Carolyn Marks,Qi Ouyang,Xinzheng Zhang,Youdong Mao,Youdong Mao,Hao Wu,Hao Wu +23 more
TL;DR: It is found that the PrgJ-NAIP2-NLRC4 inflammasome is assembled into multisubunit disk-like structures through a unidirectional adenosine triphosphatase polymerization, primed with a single PrGJ-activated NAIP2 per disk.
Journal ArticleDOI
Nanobodies as therapeutics: big opportunities for small antibodies
TL;DR: How Nbs are being explored as therapeutics in many fields of medicine, including oncology, inflammatory, infectious and neurological diseases, and imaging, is discussed and their potential for use in the diagnosis and monitoring of diseases is highlighted.
Journal ArticleDOI
Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape.
Paul-Albert Koenig,Hrishikesh Das,Hejun Liu,Beate M. Kümmerer,Florian N. Gohr,Lea-Marie Jenster,Lisa D. J. Schiffelers,Yonas M. Tesfamariam,Miki Uchima,Jennifer Deborah Wuerth,Karl Gatterdam,Natalia Ruetalo,Maria H Christensen,Caroline I. Fandrey,Sabine Normann,Jan M. P. Tödtmann,Steffen Pritzl,Leo Hanke,Jannik Boos,Meng Yuan,Xueyong Zhu,Jonathan L. Schmid-Burgk,Hiroki Kato,Michael Schindler,Ian A. Wilson,Matthias Geyer,Kerstin U. Ludwig,B. Martin Hallberg,Nicholas C. Wu,Florian I. Schmidt +29 more
TL;DR: In this paper, the authors used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes of the SARS-CoV-2 spike protein.
Journal ArticleDOI
Targeting the N terminus for site-selective protein modification.
TL;DR: This Perspective provides an overview of N-terminal modification techniques and the chemical rationale governing each, along with their uses in a number of diverse biological applications.
References
More filters
Journal ArticleDOI
Making and breaking peptide bonds: protein engineering using sortase.
TL;DR: A brief overview of the biology of sortase enzymes and current applications in protein engineering is provided and areas that lend themselves to further innovation and that suggest new applications are identified.
Journal ArticleDOI
Sortase enzymes in Gram‐positive bacteria
TL;DR: This work reviews what is known about the functions of sortase enzymes and the molecular basis of catalysis and places particular emphasis on ‘pilin’ specific class C sortases that construct structurally complex pili.
Journal ArticleDOI
Site-Specific N- and C-Terminal Labeling of a Single Polypeptide Using Sortases of Different Specificity
John M. Antos,Guo-Liang Chew,Carla P. Guimaraes,Nicholas C. Yoder,Gijsbert M. Grotenbreg,Maximilian Wei-Lin Popp,Hidde L. Ploegh +6 more
TL;DR: The generality of N-terminal labeling with SrtAstaph is demonstrated by near-quantitative labeling of multiple protein substrates with excellent site specificity.
Journal ArticleDOI
Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin
Mark Howarth,Alice Y. Ting +1 more
TL;DR: This protocol describes a simple and efficient way to label specific cell surface proteins with biophysical probes on mammalian cells through the optimal use of BirA and streptavidin for site-specific labeling and also how to produceBirA and monovalent strePTavidin.
Journal ArticleDOI
Site-specific chemical protein conjugation using genetically encoded aldehyde tags
David Rabuka,Jason S. Rush,Gregory W. Dehart,Peng Wu,Carolyn R. Bertozzi,Carolyn R. Bertozzi +5 more
TL;DR: This protocol outlines both the generation and the analysis of proteins aldehyde-tagged at their termini and the methods for chemical conjugation to the formylglycine.