Expression and purification of the chloroplast putative nitrogen sensor, PII, of Arabidopsis thaliana.
The bacterial PII protein was discovered over 30 years ago and is known to be a key player in orchestrating the coordination of nitrogen metabolism with changes in carbon flux. Bacterial PII is regulated by covalent modification and binding to effector molecules in response to the nitrogen/carbon status of the cell and appropriately coordinates the activity of glutamine synthetase and the transcription of a nitrogen sensitive regulon. Recently, a PII protein was identified in higher plants and the protein was found to be localized to the chloroplast. The Arabidopsis thaliana putative nitrogen sensor protein, PII, was cloned and overexpressed with a C-terminal 6-histidine tag. The full-length protein, which included the chloroplast transit peptide, was overexpressed in Escherichia coli, but was very susceptible to proteolytic degradation. Removal of the transit peptide yielded a highly pure, stable recombinant protein whose identity was established as PII by matrix assisted laser desorption ionization-time of flight mass spectrometry. Polyclonal antibodies generated against the recombinant protein effectively immunoprecipitated PII from an A. thaliana extract and the protein was confirmed to be 17 kDa in mass. The availability of milligram amounts of PII will allow a complete biophysical characterization of the protein and antibodies should aid in the identification of PII interacting proteins and the establishment of the higher plant PII signal transduction cascade.
Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada T2N 1N4.
Catherine S
CS
Smith
Steven T
ST
Zaplachinski
Douglas G
DG
Muench
Greg B G
GB
Moorhead
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D017360:Arabidopsis / Q000166:cytology / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000737:chemistry; D002736:Chloroplasts / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism*; D051922:PII Nitrogen Regulatory Proteins; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000906:Antibodies; D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D051922:PII Nitrogen Regulatory Proteins; C473074:PII protein, Arabidopsis; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C404143:PIID regulatory protein, Bacteria; D009584:Nitrogen