Heat stability of Proteobacterial PII protein facilitate purification using a single chromatography step

• Published in: Protein expression and purification 2012-01, Vol.81 (1), p.83-88
• Main Authors: Moure, Vivian R., Razzera, Guilherme, Araújo, Luíza M., Oliveira, Marco A.S., Gerhardt, Edileusa C.M., Müller-Santos, Marcelo, Almeida, Fabio, Pedrosa, Fabio O., Valente, Ana P., Souza, Emanuel M., Huergo, Luciano F.
• Format: Article
• Language: English
• Subjects: Azospirillum brasilense - enzymology; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Chromatography, Affinity - methods; Escherichia coli - genetics; Escherichia coli - metabolism; Heat treatment; Melting temperature; Nuclear Magnetic Resonance, Biomolecular; PII Nitrogen Regulatory Proteins - chemistry; PII Nitrogen Regulatory Proteins - isolation & purification; PII Nitrogen Regulatory Proteins - metabolism; PII proteins; Protein Conformation; Protein Multimerization; Protein NMR; Protein Stability; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Transition Temperature
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2011.09.008

► We examined how PII protein folding is affected by heat-treatment. ► We observed that Proteobacteria PII have high melting temperatures. ► We observed that PII structure and activity did not alter after a heat-treatment. ► We conclude that heat-treatment can be applied to improve PII protein purification. The PII 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 PII proteins from Azospirillum brasilense, GlnB and GlnZ, do not alter their migration behavior under native gel electrophoresis following incubated for a few minutes at 95°C. This data suggested that PII 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 1H NMR to show that the A. brasilense GlnB is stable up to 70°C. The melting temperature (Tm) of GlnB was determined to be 84°C using the fluorescent dye Sypro-Orange. PII proteins from other Proteobacteria also showed a high Tm. We exploited the thermo stability of PII by introducing a thermal treatment step in the PII purification protocol, this step significantly improved the homogeneity of A. brasilense GlnB and GlnZ, Herbaspirillum seropedicae GlnB and GlnK, and of Escherichia coli GlnK. Only a single chromatography step was necessary to obtain homogeneities higher than 95%. NMR1Abbreviations used: NMR, nuclear magnetic resonance; 2-OG, 2-oxoglutarate.1 and in vitro uridylylation analysis showed that A. brasilense GlnB purified using the thermal treatment maintained its folding and activity. The purification protocol described here can facilitate the study of PII protein family members.