Cloning of phosphatase I gene from a psychrophile, Shewanella sp., and some properties of the recombinant enzyme.
Psychrophilic phosphatase I from Shewanella sp. is a cold enzyme that was found as a novel protein-tyrosine-phosphatase (PTPase, EC 3. 1.3.48) with a histidine as its catalytic residue [Tsuruta and Aizono (1999) J. Biochem. 125, 690-695]. Here, we determined the nucleotide sequence of a DNA fragment (2,004 bp) containing the phosphatase I gene by cloning with polymerase chain reaction (PCR) and inverted PCR techniques. The deduced amino acid sequence, of the enzyme contained a conserved region of protein-serine/threonine-phosphatase (PPase). The 38.5 kDa-recombinant protein expressed in Escherichia coli was purified to homogeneity by glutathione-Sepharose 4B column chromatography, treatment with endoproteinase and Mono-Q column chromatography. The recombinant enzyme had a specific activity of 49.4 units and, like native psychrophilic phosphatase I, exhibited high catalytic activity at low temperature and PTPase activity.
Tsuruta
H
H
Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe, Hyogo 657-8501, Japan.
Aizono
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction / Q000379:methods; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D020592:Shewanella / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity / Q000235:genetics; D013696:Temperature
D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; C027606:L-glutamic acid-L-tyrosine copolymer; D017027:Protein Tyrosine Phosphatases