Abstract：
: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, 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 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 (1)H 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. 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 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%. NMR(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 P(II) protein family members.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Moure VR,Razzera G,Araújo LM,Oliveira MAS,Gerhardt ECM,Müller-Santos M,Almeida F,Pedrosa FO,Valente AP,Souza EM,Huergo LFdoi
10.1016/j.pep.2011.09.008subject
Has Abstractpub_date
2012-01-01 00:00:00pages
83-88issue
1eissn
1046-5928issn
1096-0279pii
S1046-5928(11)00255-5journal_volume
81pub_type
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