Proteomics-Based Evaluation of Cell Free Protein Synthesis

Cell free protein synthesis (CFPS) has the potential to produce enzymes, therapeutic agents, and other proteins while circumventing difficulties associated with in vivo heterologous expression. However, the contents of the cell free extracts used to carry out synthesis are largely unknown, which hampers progress toward enhancing yield or functional activity of the target protein. Building on advances in mass spectrometry (MS)-based proteomics, we explored the utility of MS for characterizing the bacterial extracts used for transcribing and translating gene sequences into proteins as well as the products of CFPS reactions. Bottom-up proteomics experiments effectively identified over 1000 proteins in these extracts. The complete set of proteins necessary for transcription and translation were present, demonstrating the ability to define potential metabolic capabilities of the extract. Further, MS-based techniques allow characterization of the CFPS product and provide insight into the synthesis reaction and potential functional activity of the product. These features were demonstrated using two different CFPS products, the commonly used standard GFP (27 kDa) and the polyketide synthase DEBS1 (394 kDa). In both cases, the peptides resulting from trypsin digestion of the product are detected by MS and suggest the extent of protein synthesis. For the large, multi-domain DEBS1, premature termination of protein translation was indicated. Additionally, MS-MS analysis, as part of a conventional “bottom-up” (untargeted) proteomics workflow, identified post-translational modifications, including the chromophore in GFP and the three phosphopantetheinylation sites in DEBS1. These post-translational modifications are essential for functional activity, and the ability to identify them with mass spectrometry is valuable for judging the success of the CFPS reaction. Collectively, the application of MS-based proteomics will prove useful for advancing the application of CFPS and related techniques employing cell extracts to challenging cases.