Session Speaker

Novel Cell-Free Protein Expression System Enables Rapid Production and Interaction Analysis of Recombinant Proteins
Sergei Mureev, Oleksiy Kovtun, Uyen T.T. Nguyen and Kirill Alexandrov
Australia

Cell-free protein synthesis enables the rapid production and engineering of recombinant proteins. Existing cell-free systems display non-overlapping properties with respect to efficiency, scalability, and the ability to produce functional eukaryotic proteins. Here, we describe the development of polymeric Species-Independent Translational Sequences (SITS) that can mediate efficient cell-free protein synthesis across the evolutionary tree through bypassing the early translation initiation factors. We used these leaders to create a novel cell-free system based on the protozoan, Leishmania tarentolae, with antisense technology used to selectively suppress translation of endogenous mRNAs. In batch mode, Leishmania tarentolae extract is more efficient and yields superior-quality recombinant proteins than the commercially available eukaryotic cell-free systems. The system can be directly programmed with unpurified PCR reactions, enabling rapid generation of large protein libraries and protein variants. Further, we demonstrate that protein:protein and protein:small molecule interactions can be quantified directly in the translation mixtures using fluorescent (cross-) correlation spectroscopy.