Cell-Free Synthesis of a Functional Ion Channel in the Absence of a Membrane and in the Presence of Detergent
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- 作者：Catherine Berrier ; Kyu-Ho Park ; Saï ; d Abes ; Anne Bibonne ; Jean-Michel Betton ; and Alexandre Ghazi
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：October 5, 2004
- 年：2004
- 卷：43
- 期：39
- 页码：12585 - 12591
- 全文大小：108K
- 年卷期：v.43,no.39(October 5, 2004)
- ISSN：1520-4995
文摘
We have investigated the possibility of cell-fee synthesis of membrane proteins in the absenceof 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 
-D-maltoside, and CHAPS, were compatible with cell-free synthesis,while n-octyl -D-glucoside and deoxycholate had an inhibitory effect. In vitro synthesis in the presenceof Triton X-100 yielded milligram amounts of MscL per milliliter of lysate. Cross-linking experimentsshowed that the protein was able to oligomerize in detergents. When the purified protein was reconstitutedin liposomes and studied by the patch-clamp technique, its activity at the single-molecule level was similarto that of the recombinant protein produced in Escherichia coli. Cell-free synthesis of membrane proteinsshould prove a valuable tool for the production of membrane proteins whose overexpression in heterologoussystems is difficult.
-D-maltoside, and CHAPS, were compatible with cell-free synthesis,while n-octyl -D-glucoside and deoxycholate had an inhibitory effect. In vitro synthesis in the presenceof Triton X-100 yielded milligram amounts of MscL per milliliter of lysate. Cross-linking experimentsshowed that the protein was able to oligomerize in detergents. When the purified protein was reconstitutedin liposomes and studied by the patch-clamp technique, its activity at the single-molecule level was similarto that of the recombinant protein produced in Escherichia coli. Cell-free synthesis of membrane proteinsshould prove a valuable tool for the production of membrane proteins whose overexpression in heterologoussystems is difficult.