The complex functioning of biological and biochemical systems requires novel strategies for high-throughput study of these complex interactions.  In order to effectively and reliably study both protein interaction and function in a high-throughput manner, we utilized cell-free protein expression in combination with various amenable assays such as AlphaLISA, ELISA and FCS.

Since cell-free protein expression decreases the timescale of protein production from days to hours, the speed and efficiency of obtaining a large number of suitable DNA-constructs from genetic libraries and producing the required amounts of DNA for protein expression are the only remaining bottlenecks. We solved these problems by Gateway cloning and Rolling Circle Amplification (RCA).

To improve the throughput of the DNA-construct production, we designed custom Gateway-based destination vectors containing specific elements and various assay-appropriate tags for successful expression in a range of cell-free systems. The implementation of Gateway cloning into our pipeline makes this step suitable for DNA-construct production in a 96-well plate format.

Production of the required amounts of DNA for cell-free expression was improved by successfully incorporating the use of RCA, which decreases cost and increases throughput over the plasmid maxipreps that are normally used. The DNA yield from RCA reactions with volumes under 100 μL was sufficient for several cell-free expression reactions and could be effectively miniaturized in a 384-well plate format.

In a case study where high throughput gateway cloning, cell-free protein expression and FCS assays were employed to map the interactions of 8 separate proteins involved in a large protein complex within a week.

These alternate DNA producing methods have enabled us to fully utilize the capacity of the high throughput assays for protein function and interaction, making  the pipeline more rapid, reliable and cost effective.