We have established a platform that combines Gateway cloning, DNA amplification and cell-free protein expression to facilitate the functional analysis of large open reading frame (ORF) libraries. 

Whether it is to probe large ORFeomes for specific functions, or to screen a library of protein mutants, the challenge is cloning and expression of the proteins of interest. We implemented a high-throughput cloning strategy using dedicated Gateway vectors, giving us access to established ORF libraries and simplifying the cloning of novel mutant libraries. In order to shortcut bacterial DNA amplification, we adopted the in-vitro Rolling Circle Amplification (RCA). The method is based on isothermal amplification of circular DNA and yields up to 500 ng/μL product. It also enables a direct link from single-clone colony to protein expression just by simple fluid handling. The proteins are then expressed using a eukaryotic cell-free extract based on Leishmania tarentolae, and can be analysed straight in the translation mixture.

In our case study we apply this platform for the selection of glycoside hydrolases. We are focusing on conforming their thermostability and activity at given temperatures and pH for biotechnological application. As the substrates of these enzymes are insoluble polysaccharides, we developed a streamlined semi-quantitative assay for their screening. Finally we will validate the structural integrity of the positive candidates by smFRET. This will showcase how the throughput for larger-scale protein analysis can be improved by the implementation of our platform.