PURIFICATION, CHARACTERIZATION, AND LOCALIZATION OF SUBUNIT INTERACTION AREA OF RECOMBINANT MOUSE RIBONUCLEOTIDE REDUCTASE R1 SUBUNIT

Mammalian ribonucleotide reductase is a heterotetramer formed by the t wo non-identical homodimers proteins R1 and R2. We have succeeded in e xpressing the 90-kDa mouse R1 protein in Escherichia coli in an active , soluble form using the T7 RNA polymerase pET vector system. To avoid inclusion bodies, the bacteria were grown at 15 degrees C with minima l concentration of the inducer isopropyl-1-thio-beta-D-galactopyranosi de. After a rapid purification procedure, approximately 20 mg of pure R1 protein were obtained per liter of bacterial culture. The concentra ted R1 protein solution had a pinkish red color. Spectroscopy in combi nation with iron and labile sulfur analyses demonstrated that the colo r originated from an iron-sulfur complex. However, all attempts to dem onstrate a function of this complex have been inconclusive. A comparis on of the recombinant R1 protein with the corresponding protein purifi ed from calf thymus showed no evidence for glycosylation. Circular dic hroism spectroscopy indicated an alpha-helical content of 50%. A flexi ble COOH-terminal tail of 7 residues in the R2 protein was earlier sho wn to be essential for binding to the R1 protein. Using a peptide prot ection assay and photoaffinity labeling, we now show that the R2 prote in tail interacts with a region close to the carboxyl terminus of the R1 protein.