Fluorescent SNAP-Tag Galectin Fusion Proteins as Novel Tools in Glycobiology

Christiane   E.   Kupper; Sophia      Böcker; Hulong      Liu; Carina      Adamzyk; Julia   van de   Kamp; Tobias      Recker; Bernd      Lethaus; Willi      Jahnen-Dechent; Sabine      Neuss; Gerhard      Muller-Newen; Lothar      Elling

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.

Keywords: Galectin-1, galectin-3, SNAP-tag, fusion protein, fluorescent protein, mesenchymal stem cells.

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