An expression system for screening of proteins for glycan and protein interactions

Diana M. E. Otto, Maria A. Campanero-Rhodes, Rositsa Karamanska, Andrew K. Powell, Nicolai Bovin, Jeremy E. Turnbull, Robert A. Field, Jonathan Blackburn, Ten Feizi, Paul R. Crocker

    Research output: Contribution to journalArticle

    11 Citations (Scopus)

    Abstract

    Here we describe a versatile high-throughput expression system that permits genome-wide screening of type 1 membrane and secreted proteins for interactions with glycans and proteins using both cell-expressed and soluble forms of the expressed proteins. Based on Gateway cloning methodology, we have engineered a destination vector that directs expression of enhanced green fluorescent protein (EGFP)-tagged proteins at the cell surface via a glycosylphosphatidylinositol tail. The EGFP fusion proteins can then be cleaved with PreScission protease to release soluble forms of proteins that can be optionally biotinylated. We demonstrate the utility of this cloning and expression system for selected low-affinity membrane lectins from the siglec family of sialic acid-binding immunoglobulin-like lectins, for the glycosaminoglycan-binding proteins FGF-1 and BACE, and for the heterotypic adhesion molecules JAM-B and JAM-C. Cell-expressed proteins can be evaluated for glycan interactions using polyvalent soluble glycan probes and for protein interactions using either cells or soluble proteins. Following cleavage from the cell surface, proteins were complexed in solution and sufficient avidity was achieved to measure weak protein-glycan and weak protein-protein interactions using glycan arrays and surface plasmon resonance, respectively. (C) 2011 Elsevier Inc. All rights reserved.

    Original languageEnglish
    Pages (from-to)261-270
    Number of pages10
    JournalAnalytical Biochemistry
    Volume411
    Issue number2
    DOIs
    Publication statusPublished - 15 Apr 2011

    Keywords

    • Cloning
    • Expression
    • Lectin
    • Glycan
    • Protein
    • Carbohydrate recognition
    • Cell recognition
    • L-selectin
    • In vitro
    • Microarrays
    • Binding
    • Adhesion
    • Receptor
    • Glycosylation
    • Mechanisms

    Cite this

    Otto, D. M. E., Campanero-Rhodes, M. A., Karamanska, R., Powell, A. K., Bovin, N., Turnbull, J. E., Field, R. A., Blackburn, J., Feizi, T., & Crocker, P. R. (2011). An expression system for screening of proteins for glycan and protein interactions. Analytical Biochemistry, 411(2), 261-270. https://doi.org/10.1016/j.ab.2010.12.036