The P-II proteins comprise a family of widely distributed signal transduction proteins that integrate the signals of cellular nitrogen, carbon and energy status, and then regulate, by protein-protein interaction, the activity of a variety of target proteins including enzymes, transcriptional regulators and membrane transporters. We have previously shown that the P-II proteins from Azospirillum brasilense, GInB and GInZ, do not alter their migration behavior under native gel electrophoresis following incubated for a few minutes at 95 degrees C. This data suggested that P-II proteins were either resistant to high temperatures and/or that they could return to their native state after having been unfolded by heat. Here we used H-1 NMR to show that the A. brasilense GInB is stable up to 70 degrees C. The melting temperature (Tm) of GInB was determined to be 84 degrees C using the fluorescent dye Sypro-Orange. P-II proteins from other Proteobacteria also showed a high Tm. We exploited the thermo stability of P-II by introducing a thermal treatment step in the P-II purification protocol, this step significantly improved the homogeneity of A. brasilense GInB and GInZ, Herbaspirillum seropedicae GInB and GInK, and of Escherichia coli GInK. Only a single chromatography step was necessary to obtain homogeneities higher than 95%. NMR1 and in vitro uridylylation analysis showed that A. brasilense GInB purified using the thermal treatment maintained its folding and activity. The purification protocol described here can facilitate the study of P-II protein family members. (C) 2011 Elsevier Inc. All rights reserved.