Recombination of human galectin-3 with the carbohydrate recognition domain to achieve glycoprotein/glycopeptide enrichment

Though plant lectins have been widely used for the enrichment of glycoproteins/glycopeptides, mammalian lectins have rarely been explored for this purpose. Using the conserved sequence of the carbohydrate recognition domain (CRD) of the human galectin. 3 protein, we engineered the following two novel human galectins: Gal3C (containing one CRD) and Tetra. Gal3C (containing four tandem CRDs). Moreover, we designed a galectin affinity column that could be used for glycoprotein/glycopeptide enrichment, by taking advantage of the ability of these recombinant galectins to become immobilized onto streptavidin agarose beads. SDS. PAGE, western blotting, and mass spectrometry were used to characterize the biological features and glycoprotein/glycopeptide enrichment abilities of both these recombinant galectins. Our results showed that both Gal3C and Tetra. Gal3C predominantly enrich glycoproteins/glycopeptides and that this enrichment process has a high specificity and sensitivity. Because Tetra. Gal3C contains three more CRD structural domains, it exhibited a better ability to achieve enrichment than Gal3C. Moreover, the immobilized Gal3C and Tetra. Gal3C were successfully used for the enrichment of glycoproteins from HepG2 cells, which indicated the selective glycoprotein/glycopeptide enrichment potential of recombinant galectins in complex samples.