PURIFICATION OF HUMAN RAD51 PROTEIN BY SELECTIVE SPERMIDINE PRECIPITATION

The human Rad51 protein is a structural homolog of Escherichia coli Re cA, The exact role of human Rad51 within the cell is poorly understood but, like its bacterial and yeast homologs, hRad51 is believed to pla y a central role in homologous recombination. However, recent reports that transgenic mice lacking the RAD51 gene die early in development s uggest an additional and essential function for mammalian Rad51 in cel l proliferation or genome maintenance. In this paper we describe a sim ple and quick method for the purification of human Rad51 overproduced in E. coli. Dialysis of cell-free extracts against buffer containing l ow concentrations of spermidine result in the formation of hRad51 micr ocrystals as observed by light and electron microscopy. The crystals w ere easily redissolved in phosphate buffer and hRad51 was further puri fied to homogeneity using hydroxylapatite, affi-gel heparin and Q-seph arose chromatography. When purified by this method hRad51 is free of e ndo-and exonuclease activities and suitable for enzymological studies, Spermidine precipitation also provides a rapid method for the large s cale purification of hRad51 suitable for physical analysis. (C) 1997 E lsevier Science B.V.