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(747g) Cell-Free Protein Synthesis: A Dynamic Platform for Rapid Screening and Production of Cytotoxic Cancer Biotherapeutics

Authors: 
Salehi, S. M. A., Brigham Young University
Smith, M. T., Brigham Young University
Bundy, B. C., Brigham Young University

Cancer, the second leading cause of death in the United States, claims the lives of 7.6 million people worldwide every year. Protein-based anticancer therapeutics are promising alternatives to traditional chemotherapies. However, many such proteins pose challenges to recombinant in vivo expression, such as cytotoxicity and aggregation, which make them difficult, time-consuming and expensive to express and screen. In this work, we demonstrate how the open nature of cell-free protein synthesis (CFPS) can be harnessed to overcome these challenges. We established an E. coli-based cell-free protein synthesis method to produce and directly assay a cytotoxic anticancer therapeutic, onconase, as a model protein. This protein is difficult to express in vivo because of inclusion body formation and its ribonucleic activity which targets three main components of the translation process: mRNA, tRNA, and rRNA. We overcame these obstacles by directly adding tRNA at specific time intervals to the CFPS environment. As a result, production yield increased 56-fold to >1.8 mg per ml with more than 95% solubility. We also established an optimized method for tRNA purification, which reduced the cost of tRNA required by 13-fold.  Finally, we demonstrate that our CFPS-produced onconase can be assayed for anticancer activity directly without any prior purification.  The ability to directly assay for therapeutic function drastically reduces the time and cost required for protein engineering and selection.