Purification of galectin-1 mutants using an immobilized Galactoseβ1–4Fucose affinity adsorbent

Takeuchi, Tomoharu; Tamura, Masahito; Ishii, Nobuaki; Ishikida, Hiroki; Sugimoto, Saori; Suzuki, Daisuke; Nishiyama, Kazusa; Takahashi, Hideyo; Natsugari, Hideaki; Arata, Yoichiro

doi:10.1016/j.pep.2015.04.001

Cited by 4 publications

(7 citation statements)

References 31 publications

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“…The cells were harvested, suspended in 10 mL ethylenediaminetetraacetic acid-phosphate buffered saline (EDTA-ME-PBS) (10 mM PO 4 3− , 0.14 M NaCl, 2.7 mM KCl, 1 mM EDTA, pH 7.4 (EDTA-PBS) with 4 mM 2-mercaptoethanol), and disrupted by sonication. The debris was removed by centrifugation and the extracts were applied to a β-galactoside-immobilized column 25) and eluted with EDTA-ME-PBS containing 0.1 M lactose.…”

Section: Chemicalsmentioning

confidence: 99%

“…The extracts were applied to a β-galactosideimmobilized column 25) and eluted with EDTA-ME-PBS containing 0.1 M lactose. Recombinant Gal-2-GFP was specifically eluted with 0.1 M lactose and the eluted protein was detected as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the molecular weight of 42 kDa.…”

Section: Chemicalsmentioning

confidence: 99%

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Identification of Galectin-2–Mucin Interaction and Possible Formation of a High Molecular Weight Lattice

Tamura

Sato

Nakajima

et al. 2017

Biological & Pharmaceutical Bulletin

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Galectins comprise a group of animal lectins characterized by their specificity for β-galactosides. Galectin-2 (Gal-2) is predominantly expressed in the gastrointestinal tract and has been identified as one of the main gastric mucosal proteins that are proposed to have a protective role in the stomach. As Gal-2 is known to form homodimers in solution, this may result in crosslinking of macromolecules with the sugar structures recognized by Gal-2. In this study, we report that Gal-2 could interact with mucin, an important component of gastric mucosa, in a β-galactoside-dependent manner. Furthermore, Gal-2 and mucin could form an insoluble precipitate, potentially through the crosslinking of mucins via Gal-2 and the formation of a lattice, resulting in a large insoluble complex. Therefore, we suggest that Gal-2 plays a role in the gastric mucosa by strengthening the barrier structure through crosslinking the mucins on the mucosal surface.

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Section: Chemicalsmentioning

confidence: 99%

Section: Chemicalsmentioning

confidence: 99%

Identification of Galectin-2–Mucin Interaction and Possible Formation of a High Molecular Weight Lattice

Tamura

Sato

Nakajima

et al. 2017

Biological & Pharmaceutical Bulletin

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“…The application of multivalent tailor-made glycans, such as neo-glycoproteins, shall enable the specific fine purification of these galectins [14,15,50,61]. Defined glycans were also used for fine purification [36]. Here, the purification of mouse Gal-1 mutants with a Galβ1-4Fuc-decorated resin was more efficient than an ASF resin.…”

Section: Galectin Purification On Glycoprotein Columnsmentioning

confidence: 99%

“…In the last years, commercially available Lactosyl-Sepharose resins were applied to purify galectins such as Gal-1, Gal-2, Gal-3, Gal-4, and Gal-8 [27,29,[33][34][35]. A Galβ1,4Fuc-decorated resin was synthesized for the selective purification of Gal-1 [36]. Since multivalency can increase the affinity of galectins, multivalent natural glycoproteins can also be used to capture galectins from the crude extract.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays

Dey¹,

Palm²,

Elling³

2023

Preprint

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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.

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“…In recent years, commercially available Lactosyl-Sepharose resins were applied to purify galectins such as Gal-1, Gal-2, Gal-3, Gal-4, and Gal-8 [ 27 , 29 , 33 , 34 , 35 ]. A Galβ1,4Fuc-decorated resin was synthesized for the selective purification of Gal-1 [ 36 ]. Since multivalency can increase the affinity of galectins, multivalent natural glycoproteins can also be used to capture galectins from the crude extract.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays

2023

View full text Add to dashboard Cite

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.

show abstract

Purification of galectin-1 mutants using an immobilized Galactoseβ1–4Fucose affinity adsorbent

Cited by 4 publications

References 31 publications

Identification of Galectin-2–Mucin Interaction and Possible Formation of a High Molecular Weight Lattice

Identification of Galectin-2–Mucin Interaction and Possible Formation of a High Molecular Weight Lattice

Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays

Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays

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