Cell-free methods to produce structurally intact mammalian membrane proteins

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 γ-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.

Production of G protein‐coupled receptors in an insect‐based cell‐free system

“…Membrane protein synthesis in insect cell‐free systems has already started over 10 years ago. Although yields for soluble proteins are frequently lower compared to E.coli and wheat germ based cell‐free systems, membrane protein synthesis in insect based cell‐free systems has meanwhile reached a comparable level (Shinoda et al, ). The insect cell‐free synthesis system initially achieved membrane protein yields in the range of 5–20 μg/mL.…”

A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis

“…Composition of the cell-free protein synthesis reactions were basically the same as previously reported (31). Dialysis method (53) was performed by using a 24-well plate filled with 1 ml feeding solution per well.…”

“…6). The addition of 'N11-tag', whose sequence is similar to that of the poly-histidine affinity tag (HAT) (31,62), to the N-terminus exhibited 20-fold increase in the yield of GST-GFPS2 fusion protein (N11GST-GFPS2 in Fig. 6), suggesting that this problem could be overcome by altering the N-terminal sequence of GST.…”

Crystal structures of claudins: insights into their intermolecular interactions

“…For crystallization, the C‐terminal fragments of claudins are truncated in all of the expression constructs . The flexible C‐terminus is dispensable for forming TJ‐like strand polymers in vitro , although the majority of claudins have a PDZ‐binding motif (–YV) at the C‐terminus that is critical for binding to many kinds of scaffolding proteins in vivo as well as for proper recruitment of claudins at TJ regions .…”

“…For crystallization, the C-terminal fragments of claudins are truncated in all of the expression constructs. [6][7][8]18 The flexible C-terminus is dispensable for forming TJ-like strand polymers in vitro, 6 although the majority of claudins have a PDZ-binding motif (-YV) at the C-terminus that is critical for binding to many kinds of scaffolding proteins in vivo 3,19 as well as for proper recruitment of claudins at TJ regions. 4 In addition, none of the three claudins contained any posttranslational lipid modifications owing to the deficient mutations in the expression constructs or properties of the cell-free protein synthesis method.…”