OVEREXPRESSION AND RAPID PURIFICATION OF BIOLOGICALLY-ACTIVE YEAST PROLIFERATING CELL NUCLEAR ANTIGEN

Yeast proliferating cell nuclear antigen (yPCNA) is a cell-cycle-regul ated protein that has been shown to be required for the efficient elon gation of primed DNA templates by DNA polymerase delta in vitro. We ha ve expressed yPCNA to a high level (greater than or equal to 30% of th e total cellular protein) with and without a six-residue histidine tag at its amino-terminus. Both forms of the recombinant protein were fou nd to be biologically active and no significant differences were obser ved between the two forms. In this report we describe an efficient met hod of extraction of DNA binding proteins, such as yPCNA, overexpresse d in Escherichia coli. The presence of a (His)(6) tag on the polypepti de permitted rapid and high-yield single-step purification of the prot ein (similar to 60 mg of purified yPCNA per liter of induced cell cult ure) by immobilized metal affinity chromatography using an imidazole g radient elution procedure. The purified yPCNA was used to characterize the biological activity and tertiary structure of the protein. Chemic al crosslinking and size-exclusion FPLC studies indicated that both fo rms of the protein have a trimeric-oligomeric structure in solution. T aken together these results indicate that both forms of the recombinan t yPCNA were similar to the endogenous protein in their biochemical pr operties. The strategies presented here are designed to maximize the y ield of recombinant protein and should prove useful to the purificatio n of other recombinant TINA binding proteins. (C) 1995 Academic Press, Inc.