RecA protein rapidly crystallizes in the presence of spermidine: a valuable step in its purification and physical characterization.
The RecA protein of Escherichia coli, whether pure or in a crude cell lysate, will rapidly form small crystals (microcrystals) in the presence of low concentrations of spermidine. We describe the conditions of time, pH, and polyamine concentration over which crystallization occurs. Microcrystal formation is inhibited by concentrations of chloride over 25 mM and concentrations of phosphate or sulfate ions as low as 2 mM. Crystallization is not inhibited by high concentrations of other proteins, and the RecA protein microcrystals are easily collected by brief centrifugation. This provides a powerful purification step with high yield. Using this novel property, we prepared over 200 mg of RecA protein at least 95% pure with a single-strand DNA-dependent ATPase activity of 98% from 65 g of cells in 2-3 days. Spermidine was easily removed from the RecA protein by dialysis.
D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008854:Microscopy, Electron; D011700:Putrescine / Q000494:pharmacology; D011938:Rec A Recombinases / Q000302:isolation & purification* / Q000378:metabolism; D013095:Spermidine / Q000494:pharmacology*; D013096:Spermine / Q000494:pharmacology
D013096:Spermine; D011938:Rec A Recombinases; D013095:Spermidine; D011700:Putrescine