Screening and characterization of variant Theta-class glutathione transferases catalyzing the activation of ethylene dibromide to a mutagen
2006 (English)In: Environmental and Molecular Mutagenesis, ISSN 0893-6692, E-ISSN 1098-2280, Vol. 47, no 9, 657-665 p.Article in journal (Refereed) Published
Ethylene dibromide (EDB) is a widespread environmental pollutant and mutagen/carcinogen. Certain Theta-class glutathione transferases (GSTs), enzymes that catalyze the reaction of reduced glutathione (GSH) with electrophiles, activate EDB to a mutagen. Previous studies have shown that human GST T1-1, but not rat GST T2-2, activates EDB. We have constructed an E. coli lacZ reversion mutagenicity assay system in which expression of recombinant GST supports activation of EDB to a mutagen. Hexa-histidine N-terminal tagging of GST T1-1 results in greatly enhanced expression of the recombinant enzyme and gives a lacZ strain that shows a mutagenic response to EDB at extremely low levels ( approximately 1 ng EDB per plate). The hexa-histidine-tagged enzyme was purified in one step by Ni(2+)-affinity chromatography. We applied the lacZ mutagenicity assay to the rapid screening of a library of variant GST Theta enzymes. Sequence variants with altered catalytic activities were identified, purified, and characterized.
Place, publisher, year, edition, pages
2006. Vol. 47, no 9, 657-665 p.
Escherichia coli, Ethylene dibromide, Glutathione transferase, Hexa-histidine tagging, lacZ, Mutagenicity
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:uu:diva-83338DOI: 10.1002/em.20252ISI: 000242773100004PubMedID: 16948056OAI: oai:DiVA.org:uu-83338DiVA: diva2:111246