A Pipeline for Studying and Engineering Single-Subunit Oligosaccharyltransferases

Thapakorn Jaroentomeechai, Xiaolu Zheng, Jasmine Hershewe, Jessica C. Stark, Michael C. Jewett, Matthew P. DeLisa*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Scopus citations

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.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc
Pages55-81
Number of pages27
DOIs
StatePublished - 2017

Publication series

NameMethods in Enzymology
Volume597
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Asparagine-linked (N-linked) protein glycosylation
  • Bacterial glycoengineering
  • Cell-free glycosylation
  • Cell-free protein synthesis
  • Directed evolution
  • Enzyme engineering
  • Nanodisc technology
  • Posttranslational modification

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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