Glutathione transferases with novel active sites isolated by phage display from a library of random mutants.
Human glutathione transferase A1-1 can be expressed as a fusion protein with coat protein III of filamentous phage f1 in a form that allows selection among variant mutant forms based on specific adsorption to immobilized active-site ligands. A library of mutant enzymes differing in the active-site region was generated by random mutagenesis of ten amino acid residues involved in the binding of electrophilic substrates. Novel glutathione transferases with altered specificity for active-site ligands were isolated by adsorption of the fusion protein on the surface of phage to analogs of an electrophilic substrate. Thus, phage display of glutathione transferase affords a system for engineering novel binding specificities onto the pre-existing protein framework of the enzyme.
Widersten
M
M
Department of Biochemistry, Uppsala University, Sweden.
Mannervik
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D017912:Inovirus / Q000235:genetics*; D007527:Isoenzymes; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D013379:Substrate Specificity
D007527:Isoenzymes; D008024:Ligands; D010455:Peptides; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha