期刊
ANALYTICAL BIOCHEMISTRY
卷 464, 期 -, 页码 17-23出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2014.07.001
关键词
RTX toxin; Ion binding; Hemolytic activity; UPEC; Erythrocytes
资金
- Jurgen Manchot Graduate School Molecules of Infection
HlyA is a toxin secreted by uropathogenic Escherichia coli strains. HlyA belongs to the repeats in the toxin protein family and needs (i) a posttranslational, fatty acylation at two internal lysines by the acyltransferase HlyC and (ii) extracellular ion binding to achieve its active conformation. Both processes are not fully understood and experiments are often limited due to the low amounts of protein available. Here, we present an optimized purification protocol for the proteins involved in HlyA activation as well as a quick and nonradioactive assay for in vitro HlyA acylation. These may simplify future experiments, e.g., activity scanning and characterization of HlyA or HlyC mutants as demonstrated with single and double HlyA lysine mutants. (C) 2014 Elsevier Inc. All rights reserved.
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