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A Pipeline for Studying and Engineering Single-Subunit Oligosaccharyltransferases.

Authors
  • Jaroentomeechai, Thapakorn1
  • Zheng, Xiaolu1
  • Hershewe, Jasmine2
  • Stark, Jessica C2
  • Jewett, Michael C3
  • DeLisa, Matthew P4
  • 1 Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, United States. , (United States)
  • 2 Northwestern University, Evanston, IL, United States. , (United States)
  • 3 Northwestern University, Evanston, IL, United States; Center for Synthetic Biology, Northwestern University, Evanston, IL, United States. , (United States)
  • 4 Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, United States. Electronic address: [email protected] , (United States)
Type
Published Article
Journal
Methods in enzymology
Publication Date
Jan 01, 2017
Volume
597
Pages
55–81
Identifiers
DOI: 10.1016/bs.mie.2017.07.011
PMID: 28935112
Source
Medline
Keywords
License
Unknown

Abstract

Asparagine-linked (N-linked) protein glycosylation is one of the most abundant types of posttranslational modification, occurring in all domains of life. The central enzyme in N-linked glycosylation is the oligosaccharyltransferase (OST), which catalyzes the covalent attachment of preassembled glycans to specific asparagine residues in target proteins. Whereas in higher eukaryotes the OST is comprised of eight different membrane proteins, of which the catalytic subunit is STT3, in kinetoplastids and prokaryotes the OST is a monomeric enzyme bearing homology to STT3. Given their relative simplicity, these single-subunit OSTs (ssOSTs) have emerged as important targets for mechanistic dissection of poorly understood aspects of N-glycosylation and at the same time hold great potential for the biosynthesis of custom glycoproteins. To take advantage of this utility, this chapter describes a multipronged approach for studying and engineering ssOSTs that integrates in vivo screening technology with in vitro characterization methods, thereby creating a versatile and readily adaptable pipeline for virtually any ssOST of interest.

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