Open AccessGeneration of Ca2+-independent sortase A mutants with enhanced activity for protein and cell surface labeling
Author(s) -
HeeJin Jeong,
Gita C. Abhiraman,
Craig M. Story,
Jessica R. Ingram,
Stephanie K. Dougan
Publication year - 2017
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0189068
Subject(s) - sortase a , sortase , calcium , biochemistry , cell , mutant , chemistry , staphylococcus aureus , biophysics , in vivo , microbiology and biotechnology , biology , bacterial protein , bacteria , gene , genetics , organic chemistry
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.