Expression, purification, crystallization, and biochemical characterization of a recombinant protein phosphatase

Abstract

A protein phosphatase (PPase) from the bacteriophage λ was overexpressed in Eschericha 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 λ-PPase. The λ-PPase shares a number of kinetic and structural properties with the eukaryotic Ser/Thr phosphatases, suggesting that the λ-PPase will serve as a good model for structure-function studies. Crystallization of the recombinant purified λ-PPase yielded monoclinic crystals. The crystals diffract to 4.0 Å when exposed to synchrotron x-ray radiation.