Site-specific N-terminal labeling of proteins using sortase-mediated reactions
Christopher S. Theile,Martin D. Witte,Annet E M Blom,Lenka Kundrat,Hidde L. Ploegh,Carla P. Guimaraes +5 more
Reads0
Chats0
TLDR
This protocol describes the use of sortase-mediated reactions to label the N terminus of any given protein of interest with a variety of probes such as fluorophores, biotin or even to other proteins.Abstract:
This protocol describes the use of sortase-mediated reactions to label the N terminus of any given protein of interest. The sortase recognition sequence, LPXTG (for Streptococcus aureus sortase A) or LPXTA (for Staphylococcus pyogenes sortase A), can be appended to a variety of probes such as fluorophores, biotin or even to other proteins. The protein to be labeled acts as a nucleophile by attacking the intermediate formed between the probe containing the LPXTG/A motif and the sortase enzyme. If sortase, the protein of interest and a suitably functionalized label are available, the reactions usually require less than 3 h.read more
Citations
More filters
Journal ArticleDOI
Unified Polymerization Mechanism for the Assembly of ASC-Dependent Inflammasomes
Alvin Lu,Venkat Giri Magupalli,Venkat Giri Magupalli,Jianbin Ruan,Jianbin Ruan,Qian Yin,Qian Yin,Maninjay K. Atianand,Matthijn R. J. Vos,Gunnar F. Schröder,Katherine A. Fitzgerald,Hao Wu,Hao Wu,Edward H. Egelman +13 more
TL;DR: It is proposed that ASC-dependent inflammasomes in both families share a unified assembly mechanism that involves two successive steps of nucleation-induced polymerization.
Journal ArticleDOI
The Current State of Peptide Drug Discovery: Back to the Future?
TL;DR: An emphasis is placed on describing research efforts to overcome the inherent weaknesses of peptide drugs, in particular their poor pharmacokinetic properties, and how these efforts have been critical to the discovery, design, and subsequent development of novel therapeutics.
Journal ArticleDOI
Imaging live-cell dynamics and structure at the single-molecule level.
TL;DR: This review discusses the concepts and strategies of structural and functional imaging in living cells at the single-molecule level with minimal perturbations to the specimen.
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.
Journal ArticleDOI
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
TL;DR: 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.
References
More filters
Book
Cystic Fibrosis
TL;DR: Advances in understanding and treatment of cystic fibrosis are summarized, focusing on pulmonary disease, which accounts for most morbidity and deaths.
Journal ArticleDOI
Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides
Book
Handbook of proteolytic enzymes
TL;DR: In this paper, Serine Peptidases with a Ser/Lys Catalytic Dyad (SC) are described, as well as their relation to the Nodavirus Coat Protein.
Journal ArticleDOI
Tricine–SDS-PAGE
TL;DR: A protocol for Tricine–SDS-PAGE is described, which includes efficient methods for Coomassie blue or silver staining and electroblotting, thereby increasing the versatility of the approach.