Inorganic pyrophosphatase (PPase) from a higher plant

Abstract

Arabidopsis thaliana inorganic pyrophosphatase (AtPPA1) coding DNA (ppa1 gene) was cloned into bacterial expression vector and overproduced in E. coli cells as a His-tagged protein. The recombinant protein was purified from the bacterial lysate by two consecutive chromatographic steps: chelating chromatography on Ni2+-charged resin followed by FPLC size exclusion chromatography. The homogenous protein was submitted for crystallization. X-Ray diffraction data extending to 1.9Å resolution were collected using synchrotron radiation. The structure was solved by molecular replacement and refinement is in progress (R-factor below 20%). The structure of AtPP1 represents an alpha+beta protein fold which overlaps with other structural models for known bacterial and yeast inorganic pyrophosphatases.