Purification of human Rad51 protein by selective spermidine precipitation.
The human Rad51 protein is a structural homolog of Escherichia coli RecA. The exact role of human Rad51 within the cell is poorly understood but, like its bacterial and yeast homologs, hRad51 is believed to play a central role in homologous recombination. However, recent reports that transgenic mice lacking the RAD51 gene die early in development suggest an additional and essential function for mammalian Rad51 in cell proliferation or genome maintenance. In this paper we describe a simple and quick method for the purification of human Rad51 overproduced in E. coli. Dialysis of cell-free extracts against buffer containing low concentrations of spermidine result in the formation of hRad51 microcrystals as observed by light and electron microscopy. The crystals were easily redissolved in phosphate buffer and hRad51 was further purified to homogeneity using hydroxylapatite, affi-gel heparin and Q-sepharose chromatography. When purified by this method hRad51 is free of endo- and exonuclease activities and suitable for enzymological studies. Spermidine precipitation also provides a rapid method for the large scale purification of hRad51 suitable for physical analysis.
Baumann
P
P
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, UK.
Benson
F E
FE
Hajibagheri
N
N
West
S C
SC
D011232:Chemical Precipitation; D002853:Chromatography, Liquid / Q000379:methods; D003460:Crystallization; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008854:Microscopy, Electron; D051135:Rad51 Recombinase; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013095:Spermidine / Q000737:chemistry*
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D013095:Spermidine