Abstract
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.
Highlights
Glycan-binding proteins such as lectins are ubiquitous proteins that mediate many biological functions
The newly developed “Capture and Release” (CaRe) method was validated by purifying lectins with different specificities and from different sources
Experiments with glycoproteins demonstrate that lectins could be used as capturing agents in the CaRe method to purify a variety of glycoproteins and possibly other glycoconjugates including proteoglycans
Summary
The newly developed “Capture and Release” (CaRe) method was validated by purifying lectins with different specificities and from different sources. As indicated by the precipitation profiles (Fig. 5A, 5B), invertase and Tg strongly interacted with C. esculenta lectin in the crude extract and formed substantial amount of cross-linked precipitates We used these two glycoproteins as capturing agents to purify C. esculenta lectin. It is a high affinity multivalent ligand of mannose specific lectin ConA [35] Taking advantage of this property, ConA was used as a capturing agent to purify SBA from crude extract (Fig. 6A). Captured SBA in the precipitated complex was released by 300 mM -methyl mannose solution in 100 mM sodium acetate buffer (pH 5.2) Both ConA and SBA are tetrameric proteins with almost identical native molecular weights at pH 7.2. Experiments with glycoproteins demonstrate that lectins could be used as capturing agents in the CaRe method to purify a variety of glycoproteins and possibly other glycoconjugates including proteoglycans
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