In vitro synthesis of membrane proteins

The heterologous expression of membrane proteins often fails because these proteins, during their synthesis, have to be inserted in the membrane of the host : a saturation of the insertion sites is probably at the origin of bad folding and/or proteolysis of the proteins. To bypass this problem, it is possible to produce the MPs in the absence of membrane and in the presence of surfactants or liposomes. One of the goals of this approach is the application of in vitro synthesis to a set of eukaryotic and prokaryotic MP and particularly to model proteins such as MscL (Mechanosensitive channel of E. coli) (Park et al, 2007), the "leader peptidase" of E. coli (LEP, type I peptidase, two transmembrane helices) and GPCRs (G-Protein Coupled Receptors).

From the expression to the functional analysis of the mechanosensitive channel MscL (adapted from Park et al 2007)

Our results with the model protein MscL are encouraging, after in vitro synthesis the protein is detected in the soluble fraction and is very efficiently (~95%) and quickly (20 min) incorporated in the liposomes. The proteoliposomes have been merged to form giant vesicles, used for patch-clamp experiments. Thereby the mechanosensitive channels obtained have the same characteristics as those found in vivo in the bacteria E. coli.



Publications

  • Fluorinated and hemifluorinated surfactants as alternatives to detergents for membrane protein cell-free synthesis. K. H. Park, C. Berrier, F. Lebaupain, B. Pucci, J.-L. Popot, A. Ghazi and F. Zito. Biochem. J. 403 : 183-187 (2007)


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