title: An expression system for screening of proteins for glycan and protein interactions.
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.
authors.affiliation: Division of Cell Biology and Immunology, Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
authors.forename: Diana M E
authors.initials: DM
authors.lastname: Otto
authors.forename: Maria A
authors.initials: MA
authors.lastname: Campanero-Rhodes
authors.forename: Rositsa
authors.initials: R
authors.lastname: Karamanska
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Powell
authors.forename: Nicolai
authors.initials: N
authors.lastname: Bovin
authors.forename: Jeremy E
authors.initials: JE
authors.lastname: Turnbull
authors.forename: Robert A
authors.initials: RA
authors.lastname: Field
authors.forename: Jonathan
authors.initials: J
authors.lastname: Blackburn
authors.forename: Ten
authors.initials: T
authors.lastname: Feizi
authors.forename: Paul R
authors.initials: PR
authors.lastname: Crocker
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000737:chemistry; D002240:Carbohydrate Sequence; D015815:Cell Adhesion Molecules / Q000737:chemistry; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D037102:Lectins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D063265:Sialic Acid Binding Immunoglobulin-like Lectins; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D015815:Cell Adhesion Molecules; D037102:Lectins; D008565:Membrane Proteins; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D063265:Sialic Acid Binding Immunoglobulin-like Lectins; C485184:enhanced green fluorescent protein; D016220:Fibroblast Growth Factor 1; D049452:Green Fluorescent Proteins; D016282:Aspartic Acid Endopeptidases