Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.

ABSTRACT: Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cel

SUBMITTER: Focke PJ 

PROVIDER: S-EPMC5021318 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature