Inventors

Kazumi Shimono, Shigeyuki Yokoyama, et al.

Summary

Membrane proteins are important drug targets, and more than half of all drugs target membrane proteins. High yield production of high quality functional membrane proteins is needed for efficient drug design and preparation of antigens. Conventional in vivo expression systems based on living cells such as E. coli have several drawbacks: the overproduced proteins on a limited space of cellular membrane cause death of the cell, a poor membrane insertion in the heterologous host. On the other hand, cell-free protein synthesis technologies have other drawbacks such as membrane proteins produced are prone to aggregation in aqueous solutions because of their high hydrophobicity. High yield production of membrane proteins with intact activity has been a significant challenge.

To overcome this problem, we developed new technology for mass production of functional membrane proteins that are efficiently integrated into lipid bilayer, in a cell-free protein expression system. The problem of aggregation is efficiently circumvented by arranging lipid bilayer environment that contains surfactant and lipid.

In this technique, a reaction mixture for cell-free protein synthesis derived from E. coli is supplemented with a steroid surfactant and lipid, and the proteins and liposomes are formed simultaneously by dialysis. As dialysis proceeds, hydrophobic proteins being synthesized or just synthesized are protected by surfactant/lipid micelles and automatically integrated into the lipid bilayers. This technique provides highly efficient production of functional membrane protein (see figure).

We used this method to successfully produce many types of human-derived multipass transmembrane proteins and used these proteins not only for crystal structure analysis, but also for preparation of antigens.

This technology provides a large amount of highly pure functional multipass transmembrane proteins without compromising their activities. It is a promising technology for drug discovery, including drug design based on crystal structure analysis of membrane proteins, or preparation of antigen specific to given GPCR.

References

• 1.U.S. Patent No.8226976, European Patent Application No.08846826.9, Japanese Patent No.5704677
• 2.Shimono et al. Protein Science 18, 2160 - 2171 (2009)