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

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.

Key words:Sorting nexin 7 (SNX7), lipid overlay assay, homogeneity, expression and purification