TY - JOUR
T1 - A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases
AU - Schoborg, Jennifer A.
AU - Hershewe, Jasmine M.
AU - Stark, Jessica C.
AU - Kightlinger, Weston
AU - Kath, James E.
AU - Jaroentomeechai, Thapakorn
AU - Natarajan, Aravind
AU - DeLisa, Matthew P.
AU - Jewett, Michael C.
N1 - Funding Information:
We thank Markus Aebi for the generous gift of the antiserum against the C. jejuni glycan. We acknowledge the Cornell University Biotechnology Resource Center (BRC) for data acquisition with their LC-MS/MS data acquisition. We thank the University of Illinois at Chicago and Chicago Biomedical Consortium for use of their LC-MS/ MS instruments at the Mass spectrometry, Metabolomics & Proteomics Facility. We thank Kyle Wilcox for training in the preparation of NDs. JAS was supported by the National Science Foundation Graduate Research Fellowship, grant number DGE-1324585. This work was also supported by the Defense Threat Reduction Agency (HDTRA1-15-1 0052/P00001), the David and Lucile Packard Foundation, the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust, the Dreyfus Teacher-Scholar program, and the National Science Foundation (MCB 1413563).
Funding Information:
Defense Threat Reduction Agency, Grant number: HDTRA1-15-1 0052/P00001; Dreyfus Teacher-Scholar program; National Science Foundation Graduate Research Fellowship, Grant number: DGE-1324585; David and Lucile Packard Foundation; National Science Foundation, Grant number: MCB 1413563; The Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust
Funding Information:
We thank Markus Aebi for the generous gift of the antiserum against the C. jejuni glycan. We acknowledge the Cornell University Biotechnology Resource Center (BRC) for data acquisition with their LC-MS/MS data acquisition. We thank the University of Illinois at Chicago and Chicago Biomedical Consortium for use of their LC-MS/MS instruments at the Mass spectrometry, Metabolomics & Proteomics Facility. We thank Kyle Wilcox for training in the preparation of NDs. JAS was supported by the National Science Foundation Graduate Research Fellowship, grant number DGE-1324585. This work was also supported by the Defense Threat Reduction Agency (HDTRA1-15-1 0052/P00001), the David and Lucile Packard Foundation, the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust, the Dreyfus Teacher-Scholar program, and the National Science Foundation (MCB 1413563). JAS and JMH designed, conducted, and analyzed OST synthesis and activity experiments. JCS, TJ, and AN designed and optimized experimental conditions. WK and JEK designed, performed, and interpreted mass spectrometry experiments. MCJ and MPD directed the studies and interpreted data. JAS, JMH, and MCJ wrote the paper with assistance from JCS, WK, JEK, TJ, AN, and MPD. NOMENCLATURE B Radioactivity signal from no plasmid control (counts per minute) CL Concentration of Leucine in CFPS reaction (?M) CF Correction factor for unit conversion = 1,000 ml/L MW Molecular weight of expressed protein (?g/?mol) NL Number of Leucine residues in expressed protein U Radioactivity signal from CFPS sample that has not been TCA precipitated (counts per minute) W Radioactivity signal from CFPS sample that has been TCA precipitated (counts per minute) Yield Yield of expressed protein (?g/ml)
Publisher Copyright:
© 2017 Wiley Periodicals, Inc.
PY - 2018/3
Y1 - 2018/3
N2 - Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 420 μg/ml for the single-subunit OST enzyme, “Protein glycosylation B” (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life.
AB - Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 420 μg/ml for the single-subunit OST enzyme, “Protein glycosylation B” (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life.
KW - PglB
KW - asparagine-linked protein glycosylation
KW - cell-free protein synthesis
KW - membrane protein
KW - nanodisc
KW - oligosaccharyltransferase
KW - post-translational modification
KW - synthetic biology
UR - http://www.scopus.com/inward/record.url?scp=85039166768&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85039166768&partnerID=8YFLogxK
U2 - 10.1002/bit.26502
DO - 10.1002/bit.26502
M3 - Article
C2 - 29178580
AN - SCOPUS:85039166768
VL - 115
SP - 739
EP - 750
JO - Biotechnology and Bioengineering
JF - Biotechnology and Bioengineering
SN - 0006-3592
IS - 3
ER -