Overproduction and purification of the Mycoplasma capricolum phosphocarrier protein, HPr, of the phosphoenolpyruvate: sugar phosphotransferase system.
The gene (ptsH) for the phosphocarrier protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system from Mycoplasma capricolum was previously cloned and sequenced. We present here the results of experiments in which the ptsH gene was cloned into a vector for high level expression in Escherichia coli of the phosphocarrier protein. Conditions were developed for overproduction and purification of HPr by a two-column procedure. The purified protein, analyzed by Edman degradation and mass spectrometry, was found to have been processed by removal of the N-terminal methionine residue. Examination of the purified protein by gel electrophoresis under isoelectric focusing conditions revealed that it has an unusually high isoelectric point.
Zhu
P P
PP
National Institutes of Health, Bethesda, Maryland 20852, USA.
Lecchi
P
P
Pannell
L
L
Jaffe
H
H
Peterkofsky
A
A
D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D007526:Isoelectric Point; D013058:Mass Spectrometry / Q000379:methods; D008715:Methionine / Q000032:analysis; D008969:Molecular Sequence Data; D009174:Mycoplasma / Q000235:genetics*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D008715:Methionine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr