Throughput applications of cell-free expression systems

The open nature of cell-free expression systems enables throughput screening approaches as a rational strategy for protein expression and protocol development. Quality and
production of membrane proteins could thus be optimized by extensive modification of their direct expression environment. Cell-free expression reactions can be performed in 96well or 24well microplates by using batch or continuous exchange configurations.

We established a throughput platform for the linear as well as for the correlated screening of compounds supplied into cell-free expression reactions. In linear screens, the concentrations of compounds are evaluated in one dimension. However, the concentration optima of different compounds can often correlate with each other. Such interfering compounds can be optimized in correlated screens using both dimensions of the microplates.
At first, basic reaction compounds such as ion concentrations, energy precursors, T7 polymerase and template concentrations were optimized and an efficient
reaction protocol suitable for expression in 96well microplates was developed. Reaction condition could later be scaled up to at least 1 ml volumes without losses in yields.

The screening approach was furthermore used
in order to evaluate a number of additives belonging to different classes of chemicals for their compatibility to the cell-free system and for their potential to modulate the expression efficiencies of membrane proteins. In
addition, for the fast evaluation of expression screens, the monitor functions of several reporter proteins were analyzed. GFP fusions proofed to be only of limited value for the production of membrane proteins in the D-CF mode. The
presence of most detergents significantly reduced the folding capacity of GFP, while the fluorescence of superfolder GFP derivatives remained largely unaffected. Correlations of the folding of monitor proteins with the folding of covalently attached membrane proteins was analysed and their suitability as expression or folding monitors in the P-CF, D-CF and L-CF expression modes has been evaluated.