Year-end Sale % OFF 
Abstract
Sortase A, a calcium-dependent transpeptidase derived from Staphylococcus aureus, is used in a broad range of applications, such as the conjugation of fluorescent dyes and other moieties to proteins or to the surface of eukaryotic cells. In vivo and cell-based applications of sortase have been somewhat limited by the large range of calcium concentrations, as well as by the often transient nature of protein-protein interactions in living systems. In order to use sortase A for cell labeling applications, we generated a new sortase A variant by combining multiple mutations to yield an enzyme that was both calcium-independent and highly active. This variant has enhanced activity for both N- and C-terminal labeling, as well as for cell surface modification under physiological conditions.
Highlights
Gram-positive bacteria display proteins at their surface for uptake of nutrients and to execute functions associated with pathogenesis
We demonstrate its enhanced sortagging activity over a broad range of calcium concentrations for both protein and cell surface labeling
We combined previously described mutations to generate new, calcium-independent sortase variants. These variants are active at both 0 and 10mM CaCl2 in applications such as N and C-terminal protein labeling and protein-protein conjugation, the activity of Ca2+-independent variants was generally lower in the absence of added calcium
Summary
Gram-positive bacteria display proteins at their surface for uptake of nutrients and to execute functions associated with pathogenesis. A family of membrane-associated transpeptidases, anchor many of these proteins to the cell surface [1]. SrtA cleaves the scissile bond between threonine and glycine to yield an acyl-enzyme intermediate. This intermediate is resolved by a nucleophilic attack of an oligoglycine-linked substrate to generate a new peptide bond between the LPXT- and oligoglycine-containing molecules [1]. Borrowing the functionality of sortase from its bacterial origin presents a wide range of possible applications. SrtA can covalently and site- link fluorescent dyes, carbohydrates and other moieties to protein substrates and to the cell surface [3,4,5].
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
