Expression, purification and phosphoinositide binding specifity of recombinant human SNX7 expressed in Escherichia coli.
- Author:
Zhan FENG
;
Tingting XU
;
Jinxin XU
- Publication Type:Journal Article
- MeSH:
Escherichia coli;
metabolism;
Genetic Vectors;
Humans;
Phosphatidylinositols;
metabolism;
Recombinant Proteins;
biosynthesis;
Sorting Nexins;
biosynthesis;
Substrate Specificity
- From:
Chinese Journal of Biotechnology
2014;30(9):1436-1445
- CountryChina
- Language:Chinese
-
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-bar)snx7), 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-BAR)SNX7 reached over 95%. Dynamic light scattering (DLS) experiment indicated that (PX-BAR)SNX7 was homogeneous in solution. Lipid overlay assay showed that (PX-BAR)SNX7 can bind to PtdIns(5)P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3.
