Screening for recombinant glutathione transferases active with monochlorobimane.
A rapid and facile colony assay has been developed for catalytically active enzymes in combinatorial cDNA libraries of mutated glutathione transferases (GST), expressed in Escherichia coli. The basis of the method is the conjugation of glutathione (GSH) with the fluorogenic substrate monochlorobimane (MCB). This screening method makes it possible to isolate and characterize one recombinant clone that is active with MCB among thousands of inactive variants. Colonies containing GSTs that catalyze the conjugation of GSH with MCB display fluorescence under long-wavelength UV light. The fluorescence is visible instantly. One rat and 11 human GSTs representing four distinct enzyme classes were studied, and all except human GST T1-1 gave rise to fluorescent colonies. The colony assay based on MCB can consequently be broadly applied for identifying active GSTs both after subcloning of wild-type enzymes and in the screening of mutant libraries. Populations of bacteria expressing GSTs can also be analyzed by flow cytometry.
Eklund
Birgitta I
BI
Department of Biochemistry, Biomedical Center, Uppsala University, Box 576, Uppsala SE-751 23, Sweden.
Edalat
Maryam
M
Stenberg
Gun
G
Mannervik
Bengt
B
D000818:Animals; D003001:Cloning, Molecular; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods; D014644:Genetic Variation; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008403:Mass Screening; D009281:Naphthalenes / Q000032:analysis / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D011720:Pyrazoles / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012542:Scattering, Radiation; D012680:Sensitivity and Specificity
D018076:DNA, Complementary; D009281:Naphthalenes; D011720:Pyrazoles; D011994:Recombinant Proteins; C059597:monochlorobimane; D005982:Glutathione Transferase; D005978:Glutathione; C008348:1-menaphthyl sulfate