EXPRESSION, PURIFICATION, CRYSTALLIZATION, AND BIOCHEMICAL-CHARACTERIZATION OF A RECOMBINANT PROTEIN PHOSPHATASE

A protein phosphatase (PPase) from the bacteriophage lambda was overex pressed in Eschericha coli. The recombinant enzyme was purified to hom ogeneity 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, NR(II), was also dephosphorylated by the lambda-PPase. The lambda-PPas e shares a number of kinetic and structural properties with the eukary otic Ser/Thr phosphatases, suggesting that the lambda-PPase will serve as a good model for structure-function studies. Crystallization of th e recombinant purified lambda-PPase yielded monoclinic crystals. The c rystals diffract to 4.0 angstrom when exposed to synchrotron x-ray rad iation.