title: Self-catalyzed immobilization of GST-fusion proteins for genome-encoded biochips.
abstract: With the surge of proteomic information that has become available in recent years from genome sequencing projects, selective and robust technologies for making protein biochips have become increasingly desirable. Herein, we describe the development of small-molecule SNAr electrophiles (smSNAREs), a new class of capture probes that enables a selective, single-step immobilization for protein biochips. This enzymology-driven approach rides on the binding and catalytic mechanism of SjGST. We have designed and synthesized mechanism-based substrate analogs 3, 4, and 5 as electrophilic precursors for conjugation of glutathione S-transferase (GST) or any of its fusion proteins. Upon evaluating the conjugation of these probes to glutathione in the presence of SjGST via UV–visible spectroscopy (UV–vis) and LC-MS techniques, we found that 3, 4, and 5 were transferable to GSH. Through the anchoring of alkyne 5 as a smSNARE probe on glass surface, we demonstrate the single-step, self-catalyzed immobilization of SjGST. Fluorescence imaging quantitatively revealed an 18-fold increase in selective binding of SjGST over random orientations (due to nonspecific binding) of the protein. Binding between GST and smSNARE surface is robust and does not reverse upon adding up to 100 mM GSH. Further, a 6-fold increase in resolution for the smSNARE surface probe was observed over commonly employed commercially available GSH-epoxy surfaces. Detailed control experiments revealed insights into the reversibility of binding and catalysis of GSH to form conjugation products with 5 in the presence of the enzyme. As an application of this protein capture technology, we printed alkaloid biosynthesis enzyme, isonitrile synthase (IsnA), to result in a biochip. Because proteins bearing a GST-fusion purification tag are commonly created through the pGEX expression system, these findings show broad potential applicability to genome-wide studies and proteomic platforms.
authors.forename: Alden E
authors.initials: AE
authors.lastname: Voelker
authors.forename: Rajesh
authors.initials: R
authors.lastname: Viswanathan
mesh_terms: D055162:Biocatalysis; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D023281:Genomics; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D040081:Protein Array Analysis / Q000379:methods*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase