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Proteomics-based Tools for Evaluation of Cell-free Protein Synthesis

Publication Type
Journal Name
American Journal of Analytical Chemistry
Publication Date
Page Numbers
11443 to 11451

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 generally not characterized, which hampers progress toward enhancing yield or functional activity of the target protein. We explored the utility of mass spectrometry (MS)-based proteomics for characterizing the bacterial extracts used for transcribing and translating gene sequences into proteins as well as the products of CFPS reactions. Untargeted proteomics experiments effectively identified over 1000 proteins per reaction. 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 fcapabilities 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 were 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 untargeted proteomics workflow, identified post-translational modifications, including the chromophore in GFP, as well as partial coverage of the three phosphopantetheinylation sites in DEBS1. A hypothesis-driven approach focused on these three sites identified all three correctly modified sites in a whole-cell lysate, but with less complete coverage in CFPS reactions. 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 to challenging cases.