Galectins are β-galactosyl-binding proteins that fulfill essential physiological functions. In the biotechnological field, galectins are versatile tools, such as in the development of biomaterial coatings or the early-stage diagnosis of cancer diseases. 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.

Human milk oligosaccharides (HMOs) have been proven to play key roles in human development and health benefits. Further research requires high-purity HMOs in large quantities. While enzymatic glycan synthesis has been proven to be highly selective and efficient, it is often associated with costly enzyme production and loss of enzymes during synthesis. Immobilization of enzymes can counteract this disadvantage, making synthesis more cost-effective. In this study, we report a versatile immobilization technique for Leloir-glycosyltransferases (GTs) onto maleimide-activated agarose by employing the SpyCatcher-SpyTag technology. The five GTs: human β1,4-galactosyltransferase 1 (β4GalT), Neisseria meningitidis β1,3- N -acetylglucosaminyltransferase (LgtA), Escherichia coli O55:H7 β1,3-galactosyltransferase (WbgO), Helicobacter pylori α1,2-fucosyltransferase (FutC), and the human α1,3- N -acetylgalactosaminyltransferase GTA/R176G were equipped with SpyCatcher (SpyC), while SpyTag (SpyT) was covalently coupled to the agarose. SpyC-GTs were extensively characterized regarding their preferred buffer system and pH optimum, divalent cation, and substrate kinetics. We successfully immobilized these five SpyC-GTs onto SpyT-agarose, reaching yields between 67 and 100% while retaining their enzymatic activity. Long-term stability over 1 month was enhanced, and reusability for six reactions on three consecutive days was shown. One-pot syntheses employing immobilized SpyC-LgtA, SpyC-WbgO, and SpyC-β4GalT resulted in the primary formation of the tetrasaccharides Lacto- N -tetraose (LNT) and Lacto- N -neotetraose (LNnT). In sequential syntheses, we were able to produce the HMOs Lacto- N -triose II (LNT II), LNT, LNnT, Lacto- N -fucopentaose type I (LNFP I), Lacto- N -neofucopentaose type I (LNnFP I), and the blood group A antigen hexaoses type I and II (BGA I and II). Graphical 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.