title: Identification of human calphoglin-induced phosphoglucomutase phosphorylation in Escherichia coli.
abstract: Orthologous proteomes, universal protein networks conserved from bacteria to mammals, dictate the core functions of cells. To isolate mammalian protein sequences that interact with bacterial signaling proteins, a BLASTP genome search was performed using catalytic domains of bacterial phosphoryl-transfer enzymes as probes. A [32P]phosphoryl-transfer assay of these mammalian cDNA-expressing Escherichia coli cells was used to screen proteins retrieved from the database. Here we report that the expression of a human protein, named calphoglin, resulted in a significant increase in the phosphorylation of a 55-kDa protein in E. coli. The phosphorylation of the 55-kDa protein was acid-stable and its isoelectric point was determined to be 5.4. The 55-kDa protein was sequentially purified from an E. coli extract using three chromatography and two-dimensional polyacrylamide gel electrophoresis. Finally, the 55-kDa protein was purified 830-fold to homogeneity and the N-terminal amino acid sequence was analyzed. The sequence obtained, AIHNRAGQPAQQ, was identical to the N-terminal amino acids of E. coli phosphoglucomutase (PGM). This method may be applicable to the detection and analysis of other orthologous proteomes.
authors.affiliation: Department of Neurophysiology, Brain Research Institute, Niigata University, 1-757 Asahi-machi, Niigata 951-8585, Japan. tingi@bri.niigata-u.ac.jp
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Ingi
authors.forename: Hirotada
authors.initials: H
authors.lastname: Mori
authors.forename: Madoka
authors.initials: M
authors.lastname: Inuzuka
mesh_terms: D000595:Amino Acid Sequence; D000837:Anion Exchange Resins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D002852:Chromatography, Ion Exchange / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010733:Phosphoglucomutase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D020539:Sequence Analysis, Protein; D014157:Transcription Factors; D014162:Transfection
chemical_list: D000837:Anion Exchange Resins; C495392:CALCOCO1 protein, human; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D014157:Transcription Factors; D010733:Phosphoglucomutase