Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery

Thapakorn Jaroentomeechai; Jessica C. Stark; Aravind Natarajan; Cameron J. Glasscock; Laura E. Yates; Karen J. Hsu; Milan Mrksich; Michael C. Jewett; Matthew P. Delisa

doi:10.1038/s41467-018-05110-x

Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery

Thapakorn Jaroentomeechai, Jessica C. Stark, Aravind Natarajan, Cameron J. Glasscock, Laura E. Yates, Karen J. Hsu, Milan Mrksich, Michael C. Jewett, Matthew P. Delisa^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

166 Scopus citations

Abstract

The emerging discipline of bacterial glycoengineering has made it possible to produce designer glycans and glycoconjugates for use as vaccines and therapeutics. Unfortunately, cell-based production of homogeneous glycoproteins remains a significant challenge due to cell viability constraints and the inability to control glycosylation components at precise ratios in vivo. To address these challenges, we describe a novel cell-free glycoprotein synthesis (CFGpS) technology that seamlessly integrates protein biosynthesis with asparagine-linked protein glycosylation. This technology leverages a glyco-optimized Escherichia coli strain to source cell extracts that are selectively enriched with glycosylation components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs). The resulting extracts enable a one-pot reaction scheme for efficient and site-specific glycosylation of target proteins. The CFGpS platform is highly modular, allowing the use of multiple distinct OSTs and structurally diverse LLOs. As such, we anticipate CFGpS will facilitate fundamental understanding in glycoscience and make possible applications in on demand biomanufacturing of glycoproteins.

Original language	English (US)
Article number	2686
Journal	Nature communications
Volume	9
Issue number	1
DOIs	https://doi.org/10.1038/s41467-018-05110-x
State	Published - Dec 1 2018

Funding

We thank Judith Merritt (Glycobia, Inc.) for providing plasmid pMW07-pglΔB, Bil Clemons (California Institute of Technology) for plasmids encoding various PglB homologs, and Markus Aebi for providing plasmid pACYCpgl2 and hR6 serum used in this work. We thank Mr. Robert Sherwood from the Cornell Proteomics and Mass Spectrometry Facility for his technical assistance acquiring the LC–MS/MS raw data files. We thank Weston Kightlinger and James Kath for providing plasmid pJL1-hEPO that was used to generate the EPO variants used in this study. Finally, we thank Jasmine Hershewe for critical discussions and sharing of reagents and ideas. This work was supported by the Defense Threat Reduction Agency (GRANT11631647 to M.P.D., M.C. J., and M.M.), National Science Foundation (Grants # CBET 1159581 and CBET 1264701 both to M.P.D. and MCB 1413563 to M.P.D. and M.C.J.), the David and Lucile Packard Foundation, and the Dreyfus Teacher-Scholar program. T.J. was supported by a Royal Thai Government Fellowship. J.C.S. and C.J.G. were each supported by a National Science Foundation Graduate Research Fellowship.

ASJC Scopus subject areas

General Chemistry
General Biochemistry, Genetics and Molecular Biology
General Physics and Astronomy

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title = "Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery",

abstract = "The emerging discipline of bacterial glycoengineering has made it possible to produce designer glycans and glycoconjugates for use as vaccines and therapeutics. Unfortunately, cell-based production of homogeneous glycoproteins remains a significant challenge due to cell viability constraints and the inability to control glycosylation components at precise ratios in vivo. To address these challenges, we describe a novel cell-free glycoprotein synthesis (CFGpS) technology that seamlessly integrates protein biosynthesis with asparagine-linked protein glycosylation. This technology leverages a glyco-optimized Escherichia coli strain to source cell extracts that are selectively enriched with glycosylation components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs). The resulting extracts enable a one-pot reaction scheme for efficient and site-specific glycosylation of target proteins. The CFGpS platform is highly modular, allowing the use of multiple distinct OSTs and structurally diverse LLOs. As such, we anticipate CFGpS will facilitate fundamental understanding in glycoscience and make possible applications in on demand biomanufacturing of glycoproteins.",

author = "Thapakorn Jaroentomeechai and Stark, \{Jessica C.\} and Aravind Natarajan and Glasscock, \{Cameron J.\} and Yates, \{Laura E.\} and Hsu, \{Karen J.\} and Milan Mrksich and Jewett, \{Michael C.\} and Delisa, \{Matthew P.\}",

note = "Funding Information: We thank Judith Merritt (Glycobia, Inc.) for providing plasmid pMW07-pglΔB, Bil Clemons (California Institute of Technology) for plasmids encoding various PglB homologs, and Markus Aebi for providing plasmid pACYCpgl2 and hR6 serum used in this work. We thank Mr. Robert Sherwood from the Cornell Proteomics and Mass Spectrometry Facility for his technical assistance acquiring the LC–MS/MS raw data files. We thank Weston Kightlinger and James Kath for providing plasmid pJL1-hEPO that was used to generate the EPO variants used in this study. Finally, we thank Jasmine Hershewe for critical discussions and sharing of reagents and ideas. This work was supported by the Defense Threat Reduction Agency (GRANT11631647 to M.P.D., M.C. J., and M.M.), National Science Foundation (Grants \# CBET 1159581 and CBET 1264701 both to M.P.D. and MCB 1413563 to M.P.D. and M.C.J.), the David and Lucile Packard Foundation, and the Dreyfus Teacher-Scholar program. T.J. was supported by a Royal Thai Government Fellowship. J.C.S. and C.J.G. were each supported by a National Science Foundation Graduate Research Fellowship. Publisher Copyright: {\textcopyright} 2018 The Author(s).",

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doi = "10.1038/s41467-018-05110-x",

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AU - Jaroentomeechai, Thapakorn

AU - Stark, Jessica C.

AU - Natarajan, Aravind

AU - Glasscock, Cameron J.

AU - Yates, Laura E.

AU - Hsu, Karen J.

AU - Mrksich, Milan

AU - Jewett, Michael C.

AU - Delisa, Matthew P.

N1 - Funding Information: We thank Judith Merritt (Glycobia, Inc.) for providing plasmid pMW07-pglΔB, Bil Clemons (California Institute of Technology) for plasmids encoding various PglB homologs, and Markus Aebi for providing plasmid pACYCpgl2 and hR6 serum used in this work. We thank Mr. Robert Sherwood from the Cornell Proteomics and Mass Spectrometry Facility for his technical assistance acquiring the LC–MS/MS raw data files. We thank Weston Kightlinger and James Kath for providing plasmid pJL1-hEPO that was used to generate the EPO variants used in this study. Finally, we thank Jasmine Hershewe for critical discussions and sharing of reagents and ideas. This work was supported by the Defense Threat Reduction Agency (GRANT11631647 to M.P.D., M.C. J., and M.M.), National Science Foundation (Grants # CBET 1159581 and CBET 1264701 both to M.P.D. and MCB 1413563 to M.P.D. and M.C.J.), the David and Lucile Packard Foundation, and the Dreyfus Teacher-Scholar program. T.J. was supported by a Royal Thai Government Fellowship. J.C.S. and C.J.G. were each supported by a National Science Foundation Graduate Research Fellowship. Publisher Copyright: © 2018 The Author(s).

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N2 - The emerging discipline of bacterial glycoengineering has made it possible to produce designer glycans and glycoconjugates for use as vaccines and therapeutics. Unfortunately, cell-based production of homogeneous glycoproteins remains a significant challenge due to cell viability constraints and the inability to control glycosylation components at precise ratios in vivo. To address these challenges, we describe a novel cell-free glycoprotein synthesis (CFGpS) technology that seamlessly integrates protein biosynthesis with asparagine-linked protein glycosylation. This technology leverages a glyco-optimized Escherichia coli strain to source cell extracts that are selectively enriched with glycosylation components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs). The resulting extracts enable a one-pot reaction scheme for efficient and site-specific glycosylation of target proteins. The CFGpS platform is highly modular, allowing the use of multiple distinct OSTs and structurally diverse LLOs. As such, we anticipate CFGpS will facilitate fundamental understanding in glycoscience and make possible applications in on demand biomanufacturing of glycoproteins.

AB - The emerging discipline of bacterial glycoengineering has made it possible to produce designer glycans and glycoconjugates for use as vaccines and therapeutics. Unfortunately, cell-based production of homogeneous glycoproteins remains a significant challenge due to cell viability constraints and the inability to control glycosylation components at precise ratios in vivo. To address these challenges, we describe a novel cell-free glycoprotein synthesis (CFGpS) technology that seamlessly integrates protein biosynthesis with asparagine-linked protein glycosylation. This technology leverages a glyco-optimized Escherichia coli strain to source cell extracts that are selectively enriched with glycosylation components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs). The resulting extracts enable a one-pot reaction scheme for efficient and site-specific glycosylation of target proteins. The CFGpS platform is highly modular, allowing the use of multiple distinct OSTs and structurally diverse LLOs. As such, we anticipate CFGpS will facilitate fundamental understanding in glycoscience and make possible applications in on demand biomanufacturing of glycoproteins.

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ER -

Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery

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