(724d) Toward Development of a Scalable Cell-Free Protein Synthesis Platform From Yeast Extract

As a compliment to in vivo protein expression platforms, cell-free protein synthesis (CFPS) systems have shown remarkable utility for making protein pharmaceuticals in recent years.  The most common CFPS platforms are derived from Escherichia coli lysate and wheat germ extract (WGE).  While both systems are useful, they do have disadvantages:  E. coli due to its limited ability to carry out post-translational modifications and WGE in terms of scalability and consistency.  To overcome these disadvantages, we are developing a novel eukaryotic CFPS platform from yeast extract.  Yeast is a particularly attractive because as a model organism it has been intensely studied and therefore has a wealth of detailed knowledge of its genetics, biochemistry, and physiology.  Moreover, yeast has simple, inexpensive, and scalable methods for cultivation under well controlled conditions and is widely used for recombinant DNA protein expression in the pharmaceutical industry. Here we report on efforts to streamline and scalable extract preparation for obtaining highly active CFPS from yeast extracts.  We examine the effect of replacing glass bead lysis with high-pressure homogenization, replacing column chromatography with dialysis for desalting, removing nuclease treatment steps, and altering the growth conditions.  Our results demonstrate improved CFPS activity and, broadly, set the stage for developing a yeast CFPS platform that provides for inexpensive, productive, and scalable expression of a variety of protein pharmaceuticals.