A capture and release method based on noncovalent ligand cross-linking and facile filtration for purification of lectins and glycoproteins
Department of Chemistry; Health Research Institute
Glycan-binding proteins such as lectins are ubiquitous proteins that mediate many biological functions. To study their various biological activities and structure-function relationships, researchers must use lectins in their purest form. Conventional purification techniques, especially affinity column chromatography, have been instrumental in isolating numerous lectins and glycoproteins. These approaches, however, are time-consuming, consist of multiple steps, and often require extensive trial-and-error experimentation. Therefore, techniques that are relatively rapid and facile are needed. Here we describe such a technique, called capture and release (CaRe). The strength of this approach is rooted in its simplicity and accuracy. CaRe purifies lectins by utilizing their ability to form spontaneous noncovalently cross-linked complexes with specific multivalent ligands. The lectins are captured in the solution phase by multivalent capturing agents, released by competitive monovalent ligands, and then separated by filtration. CaRe does not require antibodies, solid affinity matrices, specialized detectors, a customized apparatus, controlled environments, or functionalization or covalent modification of reagents. CaRe is a time-saving procedure that can purify lectins even from a few milliliters of crude protein extracts. We validated CaRe by purifying recombinant human galectin-3 and five other known lectins and also tested CaRe's ability to purify glycoproteins. Besides purifying lectins and glycoproteins, CaRe has the potential to purify other glycoconjugates, including proteoglycans. This technique could also be used for nonlectin proteins that bind multivalent ligands. Given the ubiquity of glycosylation in nature, we anticipate that CaRe has broad utility.
The Journal of Biological Chemistry
Kadav, P. D.,
Dam, T. K.
A capture and release method based on noncovalent ligand cross-linking and facile filtration for purification of lectins and glycoproteins.
The Journal of Biological Chemistry,
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