Cell-free methods to produce structurally intact mammalian membrane proteins

Takehiro Shinoda, Naoko Shinya, Kaori Ito, Yoshiko Ishizuka-Katsura, Noboru Ohsawa, Takaho Terada, Kunio Hirata, Yoshiaki Kawano, Masaki Yamamoto, Taisuke Tomita, Yohei Ishibashi, Yoshio Hirabayashi, Tomomi Kimura-Someya, Mikako Shirouzu, Shigeyuki Yokoyama

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

The crystal structures of four membrane proteins, from bacteria or a unicellular alga, have been solved with samples produced by cell-free protein synthesis. In this study, for mammalian membrane protein production, we established the precipitating and soluble membrane fragment methods: membrane proteins are synthesized with the Escherichia coli cell-free system in the presence of large and small membrane fragments, respectively, and are simultaneously integrated into the lipid environments. We applied the precipitating membrane fragment method to produce various mammalian membrane proteins, including human claudins, glucosylceramide synthase, and the Î 3-secretase subunits. These proteins were produced at levels of about 0.1-1.0 mg per ml cell-free reaction under the initial conditions, and were obtained as precipitates by ultracentrifugation. Larger amounts of membrane proteins were produced by the soluble membrane fragment method, collected in the ultracentrifugation supernatants, and purified directly by column chromatography. For several proteins, the conditions of the membrane fragment methods were further optimized, such as by the addition of specific lipids/detergents. The functional and structural integrities of the purified proteins were confirmed by analyses of their ligand binding activities, size-exclusion chromatography profiles, and/or thermal stabilities. We successfully obtained high-quality crystals of the complex of human claudin-4 with an enterotoxin.

Original languageEnglish
Article number30442
JournalScientific reports
Volume6
DOIs
Publication statusPublished - Jul 28 2016
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • General

Fingerprint Dive into the research topics of 'Cell-free methods to produce structurally intact mammalian membrane proteins'. Together they form a unique fingerprint.

Cite this