Recombinant DNA and protein technologies have taken a leading role in many forms of research over the past 40
]. Recombinant proteins have found their way to the core of most biomedical research. Classical methods of protein purification can be divided into methods that utilize chemical properties of the protein including solubility [2
], physical characteristics, e.g. isoelectric points and size [3
], and those that use selective endogenous or engineered [4
] protein affinity. Of this latter category, fusion proteins have emerged as a prominent method for purification. Fusion proteins are created by appending a full length or truncated protein to a terminal region of the protein of interest. For affinity fusion proteins, the appended protein will bind to a third, typically small, molecule that is immobilized on the surface of a polymer resin. The fusion protein will selectively bind to the surface while other proteins are washed away [5
]. In column chromatography methods, protein lysates are eluted through columns that are packed with polymer particles. Alternatively, the affinity matrix can be mixed directly with the protein lysates, agitated for some amount of time, centrifuged and collected in batch selection methods. Fusion proteins are genetically tagged with a protein of known affinity. The glutathione S-transferase (GST)-protein is frequently used to tag a protein of interest because of its affinity for the reduced form of the tripeptide, glutathione [6
]. During the elution process, excess glutathione is added to remove the tagged protein from the affinity matrix.
Traditionally, glutathione is covalently linked to the surface of activated agarose beads (Sepharose®). A thiol bond is formed between the glutathione and an alipathic spacer that is linked to a hydroxyl group on the surface of the agarose bead [7
]. Since the inception of using glutathione as a capture agent for GST fusion proteins, almost all scientific supply companies carry a variation of glutathione conjugated polymeric beads, and at least one patent has been awarded [8
] for its utility in proteins. Agarose has been widely used for glutathione conjugation. The chemistry of this linkage has multiple reaction and purification steps that restrict in-house production of these beads to chemistry laboratories [4
Thiol-ene “click” chemistry has been shown to reproducibly form covalent thioether bonds between thiol and alkene-containing molecules. Glutathione (GSH) has been covalently linked to alkene groups in poly (N-isopropyl acrylamide) (PNIPAm) polymers [9
]. While glutathione linkage to PNIPAm was confirmed, a detailed validation of the interaction with GST tagged proteins was not presented.
Using a modified form of thiol-ene chemistry and readily available materials, we demonstrate a simple, one-step method for creating gel homogenates and beads with affinity toward GST tagged proteins. Specifically, our method employs thiol-ene addition of glutathione to low molecular weight poly(ethylene glycol) diacrylate (PEGDA). Under standard conditions, the resulting polymer forms hydrogels with typical radical initiators. These gels were readily homogenized, washed, and used to purify soluble GST proteins. In our proof of concept study, GST-fused, red shifted green fluorescent protein (rsGFP) was purified with PEGDA:GSH homogenates. We also demonstrate a novel method for creating glutathione-laden PEGDA microspheres using reverse-phase emulsion polymerization. Like the homogenates, PEGDA:GSH microspheres exhibited affinity to GST-GFP and can be used to purify the protein. Either of these methods can be implemented in almost any laboratory using readily available, inexpensive reagents.