Repair of imidazole ring-opened purines in DNA: overproduction of the formamidopyrimidine-DNA glycosylase of Escherichia coli using plasmids containing the fpg+ gene

Abstract

The formamidopyrimidine-DNA glycosylase (Fapy-DNA glycosylase) of Escherichia coli (E. coli) was overexpressed by cloning the fpg+ gene on a multicopy plasmid and placing this gene under the control of the lac promoter. The lac promoter contributed significantly to the overall expression of the fpg gene only after the deletion of an inverted repeat sequence located immediately upstream from the fpg promoter. The biological purpose of the inverted repeat sequence may be associated with the termination of an adjacent gene transcribed in the same direction as the fpg gene in E. coli. Cells harboring the fpg gene under the control of the lac promoter were able to produce the Fapy-DNA glycosylase as at least 17% of the total soluble proteins. Such strains allow the preparation of milligram quantities of pure protein for use in the study of its catalytic properties and three dimensional crystal structure.