Expression, purification, crystallization, and biochemical characterization of a recombinant protein phosphatase.
A protein phosphatase (PPase) from the bacteriophage lambda was overexpressed in Escherichia coli. The recombinant enzyme was purified to homogeneity yielding approximately 17 mg of enzyme from a single liter of bacterial culture. Biochemical characterization of the enzyme showed that it required Mn2+ or Ni2+ as an activator. The recombinant enzyme was active toward serine, threonine, and tyrosine phosphoproteins and phosphopeptides. Surprisingly, the bacterial histidyl phosphoprotein, NRII, was also dephosphorylated by the lambda-PPase. The lambda-PPase shares a number of kinetic and structural properties with the eukaryotic Ser/Thr phosphatases, suggesting that the lambda-PPase will serve as a good model for structure-function studies. Crystallization of the recombinant purified lambda-PPase yielded monoclinic crystals. The crystals diffract to 4.0 A when exposed to synchrotron x-ray radiation.
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
S
S
Zhuo
J C
JC
Clemens
D J
DJ
Hakes
D
D
Barford
J E
JE
Dixon
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D002413:Cations, Divalent; D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D010749:Phosphoprotein Phosphatases