epsin2蛋白质昆虫细胞纯化及液-液相分离功能探索  

Eukaryotic purification and liquid-liquid phase separation function of epsin2 protein

作　　者：赵冰 李卓欣 李亭亭 陈肖华 李卫华[1] 李腾[1] ZHAO Bing;LI Zhuo-xin;LI Ting-ting;CHEN Xiao-hua;LI Wei-hua;LI Teng(National Center of Biomedical Analysis,Beijing 100850,China)

机构地区：[1]国家生物医学分析中心,北京100850

出　　处：《军事医学》2021年第3期208-213,共6页Military Medical Sciences

基　　金：国家自然科学基金面上项目(31871390);国家自然科学基金优秀青年科学基金项目(81922051)。

摘　　要：目的利用昆虫杆状病毒表达系统得到epsin2蛋白质并进行纯化后,探索其能否在体外发生液-液相分离。方法构建pmCherryC1-epsin2载体,在HeLa细胞内过表达后观察其胞内状态。构建pFastBac HT-B-mCherryepsin2蛋白质纯化载体,转染昆虫细胞Sf9,获得His-mCherry-epsin2融合蛋白质。进一步探索epsin2蛋白质能否在体外发生液-液相分离以及不同蛋白质浓度、盐浓度等对其的影响。结果 PONDR数据库分析发现,epsin2 C端有明显的低复杂无序区域。HeLa细胞内过表达pmCherryC1-epsin2后发现epsin2蛋白质在细胞质呈点状聚集。pFastBac HT-B-mCherry-epsin2蛋白质纯化载体构建成功后,通过转染Sf9细胞获得His-mCherry-epsin2融合蛋白质。经过镍柱和快速蛋白质液相色谱(fast protein liquid chromatography,FPLC)纯化获得高纯度的His-mCherry-epsin2蛋白质,通过宽场荧光高分辨率活细胞成像系统观察发现mCherry-epsin2蛋白质发生了浓度依赖的液-液相分离,且可受高盐及1,6-HD抑制。结论成功纯化出His-mCherry-epsin2融合蛋白质并发现其在体外可以发生液-液相分离,为后续探索epsin2如何通过相分离参与网格蛋白介导内吞(clathrin-mediated endocytosis,CME)调节生理功能奠定了基础。Objective To obtain epsin2 from baculovirus expression vector systems(BEVS)and to explore the possibility of liquid-liquid phase separation(LLPS)of epsin2 in vitro. Methods A pmCherryC1-epsin2 vector was constructed and overexpressed in HeLa cells. After the construction of the pFastBacHTB-mCherry-epsin2 vector and Blue-white screening,His-mCherry-epsin2 fusion protein was obtained by transfecting Sf9 cells. Next,the possibility of LLPS of epsin2 in vitro and such influencing factors as different protein concentrations,salt ion concentrations and small molecular compounds were studied. Results The analysis of PONDR database showed that there were obvious low complex and disordered regions in the C terminal of epsin2. After overexpression of pmCherryC1-epsin2 in HeLa cells,it was found that epsin2 presented obvious foci structure in the cytoplasm. The constructed pFastBacHTB-mCherry-epsin2 vector was then transfected into Sf9 cells. High purity His-mCherry-epsin2 fusion protein was obtained by purification with nickel column(Ni-NTA)and fast protein liquid chromatography(FPLC). Through Delta Vision wide-wield microscopy observation,it was found that mCherry-epsin2 was driven in a concentration-dependent LLPS which could be inhibited by high salt and 1,6-HD. Conclusion His-mCherry-epsin2 protein is successfully purified. epsin2 could undergo LLPS in vitro,which can contribute to subsequent exploration of how epsin2 participates in the regulation of clathrin-mediated endocytosis through phase separation.

关 键 词：epsin2 网格蛋白 内吞 真核表达 蛋白质纯化 液-液相分离 

分 类 号：S476.13[农业科学—农业昆虫与害虫防治]