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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
biochemistry, genetics and molecular biology
An expression system for screening of proteins for glycan and protein interactions
Analytical Biochemistry, Volume 411, No. 2, Year 2011
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Description
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. © 2011 Elsevier Inc. All rights reserved.
Available Materials
https://efashare.b-cdn.net/share/pmc/articles/PMC3740237/bin/mmc1.doc
Authors & Co-Authors
Otto, Diana M.E.
United Kingdom, Dundee
University of Dundee
India, Chennai
Lifeline Institute of Regenerative Medicine
Campanero-Rhodes, Maria A.
United Kingdom, London
Imperial College London
Spain, Madrid
Centro de Investigación Biomédica en Red de Enfermedades Respiratorias
Karamanska, Rositsa
United Kingdom, Norwich
John Innes Centre
Powell, Andrew K.
United Kingdom, Liverpool
University of Liverpool
United Kingdom, Liverpool
Liverpool John Moores University
Bovin, Nicolaï Vladimirovich
Russian Federation, Moscow
Shemyakin-ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences
Turnbull, Jeremy
United Kingdom, Liverpool
University of Liverpool
Field, Rob A.
United Kingdom, Norwich
John Innes Centre
Blackburn, Jonathan M.
South Africa, Cape Town
University of Cape Town
Feizi, Ten
United Kingdom, London
Imperial College London
Crocker, Paul R.
United Kingdom, Dundee
University of Dundee
Statistics
Citations: 13
Authors: 10
Affiliations: 9
Identifiers
Doi:
10.1016/j.ab.2010.12.036
ISSN:
00032697
e-ISSN:
10960309