Fluorescent in situ folding control for rapid optimization of cell-free membrane protein synthesis.

ABSTRACT: Cell-free synthesis is an open and powerful tool for high-yield protein production in small reaction volumes predestined for high-throughput structural and functional analysis. Membrane proteins require addition of detergents for solubilization, liposomes, or nanodiscs. Hence, the number of parameters to be tested is significantly higher than with soluble proteins. Optimization is commonly done with respect to protein yield, yet without knowledge of the protein folding status. This approach contains a large inherent risk of ending up with non-functional protein. We show that fluorophore formation in C-terminal fusions with green fluorescent protein (GFP) indicates the folding state of a membrane protein in situ, i.e. within the cell-free reaction mixture, as confirmed by circular dichroism

SUBMITTER: Muller-Lucks A 

PROVIDER: S-EPMC3407079 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature