重组人源SNX7蛋白在大肠杆菌中的表达、纯化及磷脂结合特异性分析

Expression, purification and phosphoinositide binding specifity of recombinant human SNX7 expressed in Escherichia coli

摘要:

Sorting nexins (SNXs)是一类含有SNX-PX结构域，并在细胞内吞和内体分选运输过程中发挥重要调节作用的蛋白。SNX7是SNXs家族中的一员，含有PX结构域和BAR结构域，属于SNX-PX-BAR亚家族。斑马鱼实验表明，SNX7是在肝脏中大量表达的抗凋亡蛋白，并在胚胎肝脏的发育中发挥关键作用。为了从蛋白水平对SNX7进行研究，首先将编码人源PX-BARSNX7 (SNX7的一个片段，包含PX和BAR结构域)的cDNA片段插入到原核表达载体p28a中，再将重组质粒转化到大肠杆菌Rosseta 2 (DE3) 中诱导表达，并用亲和层析、离子交换和分子筛层析对PX-BARSNX7进行了纯化。Western blotting结果表明，亲和层析、离子交换和分子筛层析纯化后获得了高纯度的PX-BARSNX7蛋白。动态光散射实验显示PX-BARSNX7蛋白均一性良好。磷脂结合实验表明，PX-BARSNX7具有较为广泛的磷脂酰肌醇结合能力，能够与PtdIns(5)P、PtdIns(4,5)P2和PtdIns(3,4,5)P3结合。

Abstract:

Sorting nexins (SNXs) are a large group of proteins that contain Phox (PX)domain and involve in regulating endocytosis and endosome sorting. SNX7, a member of SNXs family, contains a PX domain and a BAR domain. In zebrafish, SNX7 is a liver-enriched anti-apoptotic protein and indispensible for the liver development. A fragment of SNX7 cDNA (px-barsnx7), encoding the PX domain and the BAR domain, was inserted into the expressing vector p28a, transformed into E. coli Rosseta 2 (DE3), and then induced by isopropyl β-D-1-Thiogalactopyranoside (IPTG). After affinity, ion exchange and gel filtration purification, the purity of PX-BARSNX7 reached over 95%. Dynamic light scattering (DLS) experiment indicated that PX-BARSNX7 was homogeneous in solution. Lipid overlay assay showed that PX-BARSNX7 can bind to PtdIns(5)P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3.