title: Fluorescent SNAP-tag galectin fusion proteins as novel tools in glycobiology.
abstract: Galectins,β-galactoside binding proteins, function in several physiological and pathological processes. The further evaluation of these processes as well as possible applications of galectins in diagnosis and therapy has raised high scientific interest. Therefore, easy and reliable test systems are necessary. Here we present the simple and cost-efficient production of recombinant human galectins as fusion proteins with SNAP-tag and fluorescent proteins. These constructs show binding specificities and oligomerisation properties generally comparable to recombinant galectins. Their direct fluorescence signal was utilised by ELISA-type assay and flow cytometry analysis with human and ovine mesenchymal stem cells (MSC). Flow cytometry demonstrated glycan mediated binding of His6-SNAP-YFP-Gal- 3 to both MSC types, which was specifically inhibited by lactose. Moreover, directed immobilisation by SNAP-tag technology onto benzylguanine- activated sepharose was utilised to prepare galectin affinity columns for glycoprotein analysis and purification. The SNAPtag directed coupling yielded up to three-fold higher binding capacities for the glycoprotein standard asialofetuin compared to nondirected coupled galectin suggesting improved functionality following directed coupling.
authors: Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Worringer Weg 1, 52074 Aachen, Germany.
authors: Christiane E
authors: CE
authors: Kupper
authors: Sophia
authors: S
authors: Böcker
authors: Hulong
authors: H
authors: Liu
authors: Carina
authors: C
authors: Adamzyk
authors: Julia
authors: J
authors: van de Kamp
authors: Tobias
authors: T
authors: Recker
authors: Bernd
authors: B
authors: Lethaus
authors: Willi
authors: W
authors: Jahnen-Dechent
authors: Sabine
authors: S
authors: Neuss
authors: Gerhard
authors: G
authors: Müller-Newen
authors: Lothar
authors: L
authors: Elling
mesh_terms: D001212:Asialoglycoproteins / Q000737:chemistry; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D060748:Fetuins / Q000737:chemistry; D037161:Galectins / Q000737:chemistry*; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry*; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D057054:Molecular Imaging / Q000379:methods*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry*; D014168:Transferrin / Q000737:chemistry; D000097573:Red Fluorescent Protein
chemical_list: D001212:Asialoglycoproteins; D060748:Fetuins; D037161:Galectins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D014168:Transferrin; D000097573:Red Fluorescent Protein; C017029:asialofetuin