title: Regioselective covalent immobilization of catalytically active glutathione S-transferase on glass slides.
abstract: The high selectivity of protein farnesyltransferase was used to regioselectively append farnesyl analogues bearing bioorthogonal alkyne and azide functional groups to recombinant Schistosoma japonicum glutathione S-transferase (GSTase) and the active modified protein was covalently attached to glass surfaces. The cysteine residue in a C-terminal CVIA sequence appended to N-terminally His(6)-tagged glutathione S-transferase (His(6)-GSTase-CVIA) was post-translationally modified by incubation of purified protein or cell-free homogenates from E. coli M15/pQE-His(6)-GSTase-CVIA with yeast protein farnesyltransferase (PFTase) and analogues of farnesyl diphosphate (FPP) containing ω-azide and alkyne moieties. The modified proteins were added to wells on silicone-matted glass slides whose surfaces were modified with PEG units containing complementary ω-alkyne and azide moieties and covalently attached to the surface by a Cu(I)-catalyzed Huisgen [3 + 2] cycloaddition. The wells were washed and assayed for GSTase activity by monitoring the increase in A(340) upon addition of 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GT). GSTase activity was substantially higher in the wells spotted with alkyne (His(6)-GSTase-CVIA-PE) or azide (His(6)-GSTase-CVIA-AZ) modified glutathione-S-transferase than in control wells spotted with farnesyl-modified enzyme (His(6)-GSTase-CVIA-F).
authors: Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
authors: Rajesh
authors: R
authors: Viswanathan
authors: Guillermo R
authors: GR
authors: Labadie
authors: C Dale
authors: CD
authors: Poulter
mesh_terms: D000818:Animals; D055162:Biocatalysis; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D005898:Glass / Q000737:chemistry*; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012549:Schistosoma japonicum / Q000201:enzymology; D013237:Stereoisomerism; D013499:Surface Properties
chemical_list: D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D005982:Glutathione Transferase