1 research outputs found
Proteomics-Based Tools for 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 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.
Full proteome experiments identified over 1000 proteins per reaction.
The complete set of proteins necessary for transcription and translation
were found, demonstrating the ability to define potential metabolic
capabilities of the extract. Further, MS-based techniques allowed
characterization of the CFPS product and provided insight into the
synthesis reaction and potential functional activity of the product.
These capabilities were demonstrated using two different CFPS products,
the commonly used standard green fluorescent protein (GFP, 27 kDa)
and the polyketide synthase DEBS1 (394 kDa). For the large, multidomain
DEBS1, substantial premature termination of protein translation was
observed. Additionally, MS/MS analysis, as part of a conventional
full proteomics workflow, identified post-translational modifications,
including the chromophore in GFP, as well as the three phosphopantetheinylation
sites in DEBS1. A hypothesis-driven approach focused on these three
sites identified that all were correctly modified for DEBS1 expressed
in vivo but with less complete coverage for protein expressed 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 use of MS-based proteomics will prove advantageous for advancing
the application of CFPS and related techniques