IQUIR   05412
INSTITUTO DE QUIMICA ROSARIO
Unidad Ejecutora - UE
artículos
Título:
Regioselective Covalent Immobilization of Catalytically Active Glutathione S-Transferase on Glass Slides
Autor/es:
VISWANATHAN, R.; LABADIE, G. R.; POULTER, C. D.
Revista:
BIOCONJUGATE CHEMISTRY
Editorial:
AMER CHEMICAL SOC
Referencias:
Lugar: Washington; Año: 2013 vol. 24 p. 571 - 577
ISSN:
1043-1802
Resumen:
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 His6-tagged glutathione S-transferase (His6-GSTase-CVIA) was post-translationally modified by incubation of purified protein or cell-free homogenates from E. coli M15/pQE-His6-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 A340 upon addition of 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GT). GSTase activity was substantially higher in the wells spotted with alkyne (His6-GSTase-CVIA-PE) or azide (His6-GSTase-CVIA-AZ) modified glutathione-S-transferase than in control wells spotted with farnesyl-modified enzyme (His6-GSTase-CVIA-F).