A Pichia pastoris Cell-Free Protein Synthesis Platform

Cell-free protein synthesis is an in vitro transcription and translation process that takes advantage of the fact that cell growth is dissociated from protein production. After cell cultures have grown to the optimal cell density, the cells are lysed while preserving the cellular machinery so that they can be used in an in vitro protein synthesis reaction. The system uses three main components: cell lysate, a reaction mix, and plasmid DNA. Protein production can be performed in hours on a lab bench, expanding the possibility to evaluate multiple plasmids at once without the need to transform and select positively expressing clones.The use of Komagataella phaffii to generate the lysate for cell-free protein synthesis has been shown with a number of different strains, including a ribosome-overexpressing strain, FHL1. The high cell densities result in a highly active lysate that has been shown to be capable of producing a variety of products, including human serum albumin and virus-like particles (VLPs). Furthermore, as a eukaryotic platform, the system is capable of performing post-transcriptional modifications such as creating disulfide bonds without the need for additional folding chaperones or supplements. Here, we describe the methods required for generating cell lysate, creating a reaction mix that has been optimized through design of experiments, optimizing the concentration of DNA template, and using a reporter protein to monitor expression yields. Harnessing the potential of CFPS using K. phaffii can lead to faster prototyping of vectors before strain construction, facilitating the rapid identification of optimal protein variants. It can also be used to produce proteins that are toxic to living cells, providing an alternative source of proteins for experimentation.