Crystallization and initial X-ray crystallographic characterization of recombinant bovine inositol polyphosphate 1-phosphatase produced in Spodoptera frugiperda cells.
Bovine inositol polyphosphate 1-phosphatase, a monomeric protein with a molecular mass of 44,000 Da, hydrolyzes the 1-position phosphate from inositol 1,3,4-trisphosphate and inositol 1,4-bisphosphate. The low abundance of inositol polyphosphate 1-phosphatase in tissues has precluded structural studies requiring large quantities of enzyme. We used recombinant Baculovirus harboring the cDNA of bovine inositol polyphosphate 1-phosphatase to infect Spodoptera frugiperda (Sf9) insect cells. Recombinant protein (25 mg per 1 x 10(9) cells) was purified to homogeneity. The enzyme produced in Sf9 cells was similar to the native purified protein as determined by immunoblotting catalytic properties, and inhibition by lithium ions. Crystals of the purified recombinant enzyme were grown by vapor diffusion. Precession photography was used to determine the parameters of inositol polyphosphate 1-phosphatase crystals. The tetragonal crystals belong to the space group P4(1) or P4(3), have unit cell dimensions of a = b = 51.6 A, c = 143.3 A, alpha = beta = gamma = 90 degrees, and contain one molecule per asymmetric unit. We have collected a complete diffraction data set extending to 2.3 A and are currently attempting to solve the three-dimensional structure of bovine inositol polyphosphate 1-phosphatase using a multiple isomorphous replacement strategy.
York
J D
JD
Division of Hematology-Oncology, Washington University School of Medicine, St Louis, MO 63110.
Chen
Z W
ZW
Ponder
J W
JW
Chauhan
A K
AK
Mathews
F S
FS
Majerus
P W
PW
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004247:DNA; D008969:Molecular Sequence Data; D009036:Moths; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry
D011994:Recombinant Proteins; D004247:DNA; D010744:Phosphoric Monoester Hydrolases; C053087:inositol-1,4-bisphosphate 1-phosphatase