title: A simple enzyme-substrate localized conjugation method to generate immobilized, functional glutathione S-transferase fusion protein columns for affinity enrichment.
abstract: Immobilized protein receptors and enzymes are tools for isolating or enriching ligands and substrates based on affinity. For example, glutathione S-transferase (GST) is fused to proteins as a tag for binding to its substrate glutathione (GSH) linked to solid supports. One issue with this approach is that high-affinity interactions between receptors and ligands require harsh elution conditions such as low pH, which can result in leached receptor. Another issue is the inherent nonspecific chemical conjugation of reactive groups such as N-hydroxysuccinimide (NHS) that couple lysines to solid supports; the nonspecificity of NHS may result in residue modifications near the binding site(s) of the receptor that can affect ligand specificity. In this study, a simple conjugation procedure is presented that overcomes these limitations and results in immobilized GST fusion proteins that are functional and specific. Here, the affinity of GST for GSH was used to generate an enzyme-substrate site-specific cross-linking reaction; GSH-Sepharose was preactivated with 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) and then incubated Fc gamma receptor IIIa (FcγRIIIa)-GST. The immobilized FcγRIIIa-GST more specifically bound glycosylated immunoglobulin G1s (IgG1s) and was used to enrich nonfucosylated IgG1s from weaker binding species. This technique can be used when modifications of amino acids lead to changes in activity.
authors.affiliation: Analytical Development, Biogen, Cambridge, MA, 02142, USA; Process Biochemistry, Biogen, Cambridge, MA, 02142, USA.
authors.forename: John
authors.initials: J
authors.lastname: Coughlin
authors.affiliation: Analytical Development, Biogen, Cambridge, MA, 02142, USA.
authors.forename: Allyson
authors.initials: A
authors.lastname: Masci
authors.affiliation: Process Biochemistry, Biogen, Cambridge, MA, 02142, USA.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Gronke
authors.affiliation: Analytical Development, Biogen, Cambridge, MA, 02142, USA.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Bergelson
authors.affiliation: Analytical Development, Biogen, Cambridge, MA, 02142, USA. Electronic address: carl.co@biogen.com.
authors.forename: Carl
authors.initials: C
authors.lastname: Co
mesh_terms: D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D017452:Receptors, IgG / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry
chemical_list: D004800:Enzymes, Immobilized; C496124:FCGR3A protein, human; D007074:Immunoglobulin G; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D012685:Sepharose; D005982:Glutathione Transferase; D005978:Glutathione; D005022:Ethyldimethylaminopropyl Carbodiimide
keywords: 1-Ethyl-3-(dimethylaminopropyl)carbodiimide (EDC); Antibody; Fc gamma receptor; Glutathione S-transferase; Glutathione Sepharose; Nonfucosylation