GST pull-down结合质谱技术鉴定Muted蛋白相互作用蛋白

Identification of Muted-interacting Proteins by GST Pull-down and Mass Spectrometry

目的鉴定Muted蛋白相互作用蛋白。方法表达并纯化GST-Muted蛋白融合蛋白,与小鼠脑组织蛋白共孵育,银染获得与Muted互作的蛋白条带,利用质谱技术获得这些条带的氨基酸序列,鉴定Muted的相互作用蛋白。结果初步鉴定出31个与Muted特异结合的互作蛋白,包括外泌体蛋白、细胞骨架蛋白及马达蛋白等,进一步应用免疫共沉淀技术验证了Muted与Stxbp1的相互作用。结论从小鼠脑组织初步鉴定了Muted蛋白的结合蛋白,为进一步研究Muted蛋白在LDCVs形成和分泌过程中的功能及机制提供了新的线索。

Objective To identify muted-interacting proteins.Methods GST-muted fusion protein was purified and incubated with mouse brain tissues.Protein bands that interacted with Muted were obtained by silver staining.Mass spectrometry was used to obtain the amino acid sequence of these bands and identify Muted-interacting proteins.Results Thirty-one potential interacting partners of Muted was identified,including proteins distributed in extracellular exosome,cytoskeleton and motors.Immunoprecipitation was used to verify the interaction between Muted and Stxbp1.Conclusion The preliminary identification of the binding protein of Muted protein from mouse brain tissue provides new clues for further study of the function and mechanism of Muted protein in the formation and secretion of LDCVs.