Generation and Use of Recombinant Galectins

Abstract

Galectins are soluble carbohydrate binding proteins that can bind β-galactosecontaining glycoconjugates by means of a conserved carbohydrate recognition domain (CRD). In mammalian systems, galectins have been shown to mediate very important roles in innate and adaptive immunity as well as facilitating host-pathogen relationships. Many of these studies have relied on purified recombinant galectins to uncover key features of galectin biology. A major limitation to this approach is that certain recombinant galectins purified using standard protocols are easily susceptible to loss of glycan-binding activity. As a result, biochemical studies that employ recombinant galectins can be misleading if the overall activity of a galectin remains unknown in a given assay condition. This article examines fundamental considerations when purifying galectins by lactosyl-sepharose and nickel-NTA affinity chromatography using human galectin-4N and-7 as examples, respectively. As other approaches are also commonly applied to galectin purification, we also discuss alternative strategies to galectin purification, using human galectin-1 and-9 as examples.

Full-Length Galectin-3 Is Required for High Affinity Microbial Interactions and Antimicrobial Activity

“…Expression plasmids encoding human Gal-3 and Gal-3C were transformed into E.coli BL21 (DE3), and Gal-3 and Gal-3C were then expressed as outlined previously (Stowell et al, 2010;Wu et al, 2021b). Briefly, transformed bacteria were cultured in LB broth containing 100 μg/ml ampicillin with agitation (250 rpm) at 37°C.…”

Innate immune Galectin-7 specifically targets microbes that decorate themselves in blood group-like antigens

“…Human Gal-3 and Gal-7 were expressed as outlined previously ( Paul et al., 2022 ; Stowell et al., 2010 ). Briefly, Gal-3 or Gal-7 transformant positive E. coli BL21 (DE3) were cultured in LB broth containing 100 μg/mL ampicillin with agitation (250 rpm) at 37°C ( Paul et al., 2022 ; Wu et al., 2021c ). When bacteria were grown to the mid-log phase, protein expression was induced by addition of isopropyl 1-thio-β-D-galactopyranoside (IPTG, 1.5 mM).…”

“…Labeled Gal-3 or Gal-7 were purified again by lactosyl-sepharose column to remove possible inactive protein that may have formed during labeling process. Bound Gal-7 was eluted with 100 mM lactose in PBS plus 2-mercaptoethanol (2-ME) and then a PD-10 gel filtration column was used to remove 2-ME and lactose prior to the use of Gal-7 for experiments ( Blenda et al., 2022 ; Stowell et al., 2007 , 2008b ; Wu et al., 2021b , 2021c ). The molecular weight of monomeric Gal-7 is 15 kDa.…”

Galectin-9 recognizes and exhibits antimicrobial activity toward microbes expressing blood group–like antigens

“…Galectin-9 growth and purification: Expression plasmid encoding human full-length Gal-9 was transformed into E. coli BL21 (DE3) and was then expressed as outlined previously (61). Briefly, transformed bacteria were cultured in LB broth containing 100 μg/mL ampicillin with shaking at 250 rpm and incubated at 37 °C.…”