Trypsin Sensitivity Assay to Study the Folding Status of Proteins
Trypsin Sensitivity Assay to Study the Folding Status of Proteins
复制标题
用于研究蛋白质折叠状态的胰蛋白酶敏感性测定
DOI：
10.21769/bioprotoc.1952
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
K. Mori
中科院分区：
文献类型：
--
作者：
Satoshi Ninagawa;K. Mori
[摘要]该方法旨在利用多肽：N-聚糖酶(PNGase)的敏感性来评估蛋白质的折叠状态。在胞浆中，PNGase是一种脱糖酶。由于PNGase对非天然蛋白质的偏好，未折叠/错误折叠蛋白上的N-糖链比折叠蛋白上的N-糖链更容易受到PNGase的影响。PNGase内源性表达于多种细胞类型，包括HCT116细胞、DT40细胞和小鼠胚胎成纤维细胞。通过在SDS-PAGE中较快的条带迁移，可以检测到PNGase的部分脱糖作用。你可以比较感兴趣的野生型和突变型蛋白质的折叠紧密度。这种方法可以与常规的分子和细胞生物学设备一起使用，但只适用于糖蛋白。
[Abstract] This protocol aims to evaluate folding status of proteins, utilizing peptide:N-glycanase (PNGase) sensitivity. In the cytosol, PNGase works as a deglycosylation-enzyme. N-glycans on unfolded/misfolded proteins are more susceptible to PNGase than N-glycans on folded proteins because of the preference of PNGase to non-native proteins. PNGase is endogenously expressed in various cell types, including HCT116 cells, DT40 cells and mouse embryonic fibroblast cells. Partial deglycosylation by PNGase can be detected by faster migration of band in SDS-PAGE. You can compare tightness of the folding among wild-type and mutant proteins of interest. This method can be used with regular molecular and cell biology equipment, but applied only to glycoproteins.