Cell-Free Synthesis of a Functional Ion Channel in the Absence of a Membrane and in the Presence of Detergent

• Published in: Biochemistry (Easton) 2004-10, Vol.43 (39), p.12585-12591
• Main Authors: Berrier, Catherine, Park, Kyu-Ho, Abes, Saïd, Bibonne, Anne, Betton, Jean-Michel, Ghazi, Alexandre
• Format: Article
• Language: English
• Subjects: Cell Membrane Permeability - genetics; Cell-Free System - chemistry; Cetomacrogol; Cholic Acids; Deoxycholic Acid; Detergents; Escherichia coli Proteins - chemical synthesis; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Genetic Vectors; Glucosides; Ion Channels - chemical synthesis; Ion Channels - genetics; Ion Channels - metabolism; Lipid Bilayers - chemistry; Mechanotransduction, Cellular - genetics; Octoxynol; Patch-Clamp Techniques; Polymers - chemical synthesis; Polymers - metabolism; Polysorbates; Solubility
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi049049y

We have investigated the possibility of cell-fee synthesis of membrane proteins in the absence of a membrane and in the presence of detergent. We used the bacterial mechanosensitive channel MscL, a homopentamer, as a model protein. A wide range of nonionic or zwitterionic detergents, Triton X-100, Tween 20, Brij 58p, n-dodecyl β-d-maltoside, and CHAPS, were compatible with cell-free synthesis, while n-octyl β-d-glucoside and deoxycholate had an inhibitory effect. In vitro synthesis in the presence of Triton X-100 yielded milligram amounts of MscL per milliliter of lysate. Cross-linking experiments showed that the protein was able to oligomerize in detergents. When the purified protein was reconstituted in liposomes and studied by the patch-clamp technique, its activity at the single-molecule level was similar to that of the recombinant protein produced in Escherichia coli. Cell-free synthesis of membrane proteins should prove a valuable tool for the production of membrane proteins whose overexpression in heterologous systems is difficult.