Engineering of a sulfotyrosine-recognizing small protein scaffold for the study of protein tyrosine O-sulfation

Justin Lawrie, Wei Niu, Jiantao Guo

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Protein tyrosine O-sulfation is considered as one of the most common types of posttranslational modification of tyrosine in nature. The introduction of a negatively charged sulfate group plays crucial roles in extracellular biomolecular interactions that dictate various cellular processes, including cell adhesion, leukocyte trafficking, hormone activities, and immune responses. Despite substantial advances in our knowledge about protein tyrosine O-sulfation in recent years, our understanding of its biological significance is still in its infancy. This is largely hindered by a chronic lack of suitable biochemical tools. We seek to meet this challenge by engineering a small protein scaffold that can recognize sulfated tyrosine (sulfotyrosine) residues with high affinity. In this chapter, we describe the directed evolution of a Src Homology 2 (SH2) domain to recognize sulfotyrosine. In the first part, the design strategy for the phage display of SH2 variants is discussed. In the second part, the techniques required for phage propagation and selection are described. The evolved SH2 variants are characterized and validated in vitro through fluorescence polarization assays. Finally, the evolved SH2 domain mutants are applied to the visualization of sulfated proteins on the cell surface.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
EditorsArun K. Shukla
PublisherAcademic Press Inc.
Pages67-89
Number of pages23
ISBN (Print)9780128181195
DOIs
Publication statusPublished - 2019

Publication series

NameMethods in Enzymology
Volume622
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

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Keywords

  • Biomolecular recognition
  • Phage display
  • Protein engineering
  • Protein tyrosine O-sulfation
  • SH2 domain
  • Sulfotyrosine

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Lawrie, J., Niu, W., & Guo, J. (2019). Engineering of a sulfotyrosine-recognizing small protein scaffold for the study of protein tyrosine O-sulfation. In A. K. Shukla (Ed.), Methods in Enzymology (pp. 67-89). (Methods in Enzymology; Vol. 622). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2019.02.004