Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY, United States.
Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY, United States.
Methods Enzymol. 2020;638:109-127. doi: 10.1016/bs.mie.2020.02.017. Epub 2020 Mar 31.
The NlpC/p60-family of peptidoglycan hydrolases are key enzymes that facilitate bacterial cell division and also modulate microbe-host interactions. These endopeptidases utilize conserved Cys-His residues in their active site and are expressed in most bacterial species as well as some eukaryotes. Here we describe methods for biochemical analysis of Enterococcus faecium SagA-NlpC/p60 peptidoglycan hydrolase activity (Kim et al., 2019; Rangan et al., 2016), which includes recombinant protein preparation and biochemical analysis using both gel-based and LC-MS profiling of peptidoglycan fragments. These protocols should also facilitate the biochemical analysis of other NlpC/p60 peptidoglycan hydrolases.
NlpC/p60 家族的肽聚糖水解酶是促进细菌细胞分裂和调节微生物-宿主相互作用的关键酶。这些内切肽酶在其活性位点利用保守的 Cys-His 残基，在大多数细菌物种以及一些真核生物中表达。在这里，我们描述了粪肠球菌 SagA-NlpC/p60 肽聚糖水解酶活性的生化分析方法（Kim 等人，2019 年；Rangan 等人，2016 年），包括重组蛋白的制备和使用基于凝胶和 LC-MS 的肽聚糖片段分析的生化分析。这些方案也应该有助于其他 NlpC/p60 肽聚糖水解酶的生化分析。
