In the cauldron of cell-free synthesis of membrane proteins: playing with new surfactants.
Cell-free protein synthesis is a well-known technique for the roles it has played in deciphering the genetic code and in the beginnings of signal sequence studies. Since then, many efforts have been made to optimise this technique and, recently, to adapt it to membrane protein production with yields compatible with structural investigations. The versatility of the method allows membrane proteins to be obtained directly stabilised in surfactant micelles or inserted in a lipidic environment (proteoliposome, bicelle, and nanodisc) at the end of synthesis. Among the surfactants used, non-detergent ones such as fluorinated surfactants proved to be a good alternative in terms of colloidal stability and preservation of the integrity of membrane proteins, as shown for Escherichia coli homo-pentameric channel, MscL (Park et al., Biochem. J., 403: 183-187). Here we report cell-free expression of Escherichia coli leader peptidase (a transmembrane protease), Halobacterium salinarium bacteriorhodopsin (a transmembrane protein binding a hydrophobic cofactor) and E. coli MscL in the presence of non-detergent surfactants, amphipols and fluorinated surfactants in comparison to their expression in classical detergents. The results confirm the potentialities of fluorinated surfactants and, although pointing to limitations in using the first generations amphipols, results are discussed in the light of membrane protein refolding, especially in the case of bacteriorhodopsin. Preliminary experiments using new generations of amphipols supports choices made in developing new molecules.
Park
Kyu-Ho
KH
Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, UMR 7099, CNRS and Université Paris-7, Institut de Biologie Physico-Chimique, CNRS FRC 550, F-75005 Paris, France.
Billon-Denis
Emmanuelle
E
Dahmane
Tassadite
T
Lebaupain
Florence
F
Pucci
Bernard
B
Breyton
Cécile
C
Zito
Francesca
F
D002474:Cell-Free System / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D013501:Surface-Active Agents / Q000737:chemistry*
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D013501:Surface-Active Agents