Expression, purification and crystallization of the SKICH domain of human TAX1BP1

Abstract

TAX1BP1 is a highly conserved, pleiotropic protein that plays many essential functions in human cells, including negative regulation of inflammatory and antimicrobial responses mediated by NF-κB and IRF3 signaling, inhibition of apoptosis, transcriptional coactivation and autophagy etc. TAX1BP1 contains a SKICH domain at the N-terminus, three coiled-coil domains in the middle and two ubiquitin-binding zinc-finger motifs at the C-terminus. The SKICH domain and the linker sequence between the SKICH domain and the coiled-coil region mediate interaction with ubiquitin-like proteins of the LC3/GABARAP family, which are autophagosome markers. For structure determination of the SKICH domain of TAX1BP1, a protein construct (amino acids 15-148) corresponding to the SKICH domain plus the linker region was expressed, purified and crystallized. A native diffraction data set has been collected to 1.9 Å resolution. A molecular-replacement solution has been found by using the structure of the SKICH domain of NDP52, a paralog of TAX1BP1.

An engineered oncolytic virus expressing PD-L1 inhibitors activates tumor neoantigen-specific T cell responses

“…1d). iPDL1 protein purified from the supernatants of VV-iPDL1/ GM-infected tumor cells 30,31 was able to bind to PD-L1 + tumor cells, but not to PD-L1-knocked down tumor cells in vitro (Fig. 1e).…”

Glucose‐regulated protein 78 (GRP78) binds directly to PIP3 phosphatase SKIP and determines its localization

“…Based on its structural model, the SKIP SKICH domain is likely to have an Ig‐like 8‐stranded β‐barrel structure (Yang et al . , ). The β‐barrel protein is abundant in the outer membrane of Gram‐negative bacteria and in the mitochondria of eukaryotes (Koebnik et al .…”

Crystallographic and modelling studies suggest that the SKICH domains from different protein families share a common Ig-like fold but harbour substantial structural variations

“…Most likely, the interdomain contact does not represent a genuine protein-protein interaction but merely a result of crystal packing. Gel-filtration data indicates that the TAX1BP1 SKICH domain exists as a monomer in solution (Yang et al, 2014).…”

“…The detailed experimental procedures for the molecular cloning and preparation of the human TAX1BP1 SKICH domain protein construct has been described elsewhere (Yang, Wang, Huang, & Du, 2014). Briefly, a DNA encoding, the putative SKICH domain and an extra C-terminal sequence (residues 15-148 of human TAX1BP1, GenBank: NP_001073333) was amplified by PCR.…”