title: Cloning, expression, and purification of galectins for in vitro studies.
abstract: Galectins are best known for their ability to bind glycoconjugates containing β-galactose, but classification of these small proteins within the galectin family is also defined by amino acid homology within structural domains and exon/intron junctions within genes. As galectins are expressed by organisms as diverse as some fungi, C. elegans, fish, birds, and mammals, and biological activities attributed to galectins are equally diverse, it becomes essential to identify, clone, and characterize galectins from many sources. Glutathione S-transferase (GST) fused to the amino-terminus of galectin cDNAs has proven to be especially useful for preparation of recombinant galectins in bacteria for use on glycan arrays, in experiments with cultured or isolated cells, and in pull-down assays with immunopurified glycoproteins. Many galectins are stabilized by reducing reagents, such that binding and elution of GST-galectins from glutathione-conjugated Sepharose with excess glutathione is both efficient and innocuous. The ability to bind and elute GST-galectins from lactose-conjugated Sepharose with excess lactose provides a relatively easy means to insure that galectins are competent for glycoconjugate binding prior to experimentation. This chapter focuses primarily on the varied approaches to use GST-galectin binding to glutathione- and lactose-conjugated Sepharose to purify recombinant galectins and then develop effective experimental protocols to characterize the specificity, interactions, and function of galectins cloned from any source. We provide one example where a pull-down assay with all the GST-tagged canine galectins reveals that the C-terminal carbohydrate recognition domain of galectin-9 (Gal-9C) specifically recognizes the glycan-dependent apical targeting signal from the glycoprotein MUC1.
authors.affiliation: Renal-Electrolyte Division, Department of Medicine, Laboratory of Epithelial Cell Biology, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Poland
authors.forename: Carol L
authors.initials: CL
authors.lastname: Kinlough
authors.forename: Rebecca P
authors.initials: RP
authors.lastname: Hughey
mesh_terms: D000818:Animals; D050260:Carbohydrate Metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics; D037161:Galectins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D061985:Madin Darby Canine Kidney Cells; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D037161:Galectins; D011993:Recombinant Fusion Proteins