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Title: Proteomics-Based Tools for Evaluation of Cell-Free Protein Synthesis

Abstract

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. Here 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 wellmore » 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.« less

Authors:
ORCiD logo [1]; ORCiD logo [1];  [1];  [2];  [1];  [2]; ORCiD logo [2]; ORCiD logo [3]; ORCiD logo [2]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Chemical Sciences Division
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biology & Soft Matter Division; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Joint Inst. for Neutron Sciences, Shull Wollan Center
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1408586
DOE Contract Number:  
AC05-00OR22725
Resource Type:
Journal Article
Journal Name:
Analytical Chemistry
Additional Journal Information:
Journal Volume: 89; Journal Issue: 21; Journal ID: ISSN 0003-2700
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Hurst, Gregory B., Asano, Keiji G., Doktycz, Charles J., Consoli, Elliot J., Doktycz, William L., Foster, Carmen M., Morrell-Falvey, Jennifer L., Standaert, Robert F., and Doktycz, Mitchel John. Proteomics-Based Tools for Evaluation of Cell-Free Protein Synthesis. United States: N. p., 2017. Web. doi:10.1021/acs.analchem.7b02555.
Hurst, Gregory B., Asano, Keiji G., Doktycz, Charles J., Consoli, Elliot J., Doktycz, William L., Foster, Carmen M., Morrell-Falvey, Jennifer L., Standaert, Robert F., & Doktycz, Mitchel John. Proteomics-Based Tools for Evaluation of Cell-Free Protein Synthesis. United States. doi:10.1021/acs.analchem.7b02555.
Hurst, Gregory B., Asano, Keiji G., Doktycz, Charles J., Consoli, Elliot J., Doktycz, William L., Foster, Carmen M., Morrell-Falvey, Jennifer L., Standaert, Robert F., and Doktycz, Mitchel John. Tue . "Proteomics-Based Tools for Evaluation of Cell-Free Protein Synthesis". United States. doi:10.1021/acs.analchem.7b02555.
@article{osti_1408586,
title = {Proteomics-Based Tools for Evaluation of Cell-Free Protein Synthesis},
author = {Hurst, Gregory B. and Asano, Keiji G. and Doktycz, Charles J. and Consoli, Elliot J. and Doktycz, William L. and Foster, Carmen M. and Morrell-Falvey, Jennifer L. and Standaert, Robert F. and Doktycz, Mitchel John},
abstractNote = {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. Here 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.},
doi = {10.1021/acs.analchem.7b02555},
journal = {Analytical Chemistry},
issn = {0003-2700},
number = 21,
volume = 89,
place = {United States},
year = {2017},
month = {10}
}