PMID:15449948
| Citation |
Berrier, C, Park, KH, Abes, S, Bibonne, A, Betton, JM and Ghazi, A (2004) Cell-free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent. Biochemistry 43:12585-91 |
|---|---|
| Abstract |
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 beta-D-maltoside, and CHAPS, were compatible with cell-free synthesis, while n-octyl beta-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. |
| Links |
PubMed Online version:10.1021/bi049049y |
| Keywords |
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 |
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