Dey, C.; Palm, P.; Elling, L. Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays. Molecules2023, 28, 1054.
Dey, C.; Palm, P.; Elling, L. Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays. Molecules 2023, 28, 1054.
Dey, C.; Palm, P.; Elling, L. Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays. Molecules2023, 28, 1054.
Dey, C.; Palm, P.; Elling, L. Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays. Molecules 2023, 28, 1054.
Abstract
Galectins are β-galactosyl-binding proteins that fulfill essential physiological functions. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His6-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His6-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6-3-fold increase in binding efficiency for HSYGal-3 (His6-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His6-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His6-tagged galectins which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study.
Biology and Life Sciences, Biology and Biotechnology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
