Tartrate-resistant bone acid phosphatase: Large-scale production and purification of the recombinant enzyme, characterization, and crystallization

Tartrate-resistant acid phosphatase (TRAP) is an enzyme expressed in bone-r esorbing osteoclasts and certain tissue macrophages in human tissues. The f unctions of TRAP in biological systems are not known. Elucidation of the th ree-dimensional structure of the active site could yield important informat ion about the physiological substrate(s) of the enzyme. We have produced re combinant rat bone TRAP using a baculovirus expression vector system. The p roduction was scaled up to a 30-1 bioreactor, and a method of purification in large scale was developed. The enzyme is composed of one 34 kDa polypept ide chain. Trypsin digestion resulted ina preparation where two subunits of similar to 23 kDa and similar to 16 kDa appeared after disulfide reduction . Trypsin digestion activated the enzyme. We generated monoclonal antibodie s against recombinant TRAP. One of the selected antibodies detected the 23 kDa subunit in Western blotting. The reduced and oxidized forms of the enzy me could be separated by Mono-S cation-exchange chromatography, Crystals of TRAP have been obtained with ammonium sulfate/ polyethylene glycol as prec ipitant. They belong to space group P2(1)2(1)2(1) or P2(1)2(1)2 with unit c ell dimensions a = 57.2 Angstrom, b = 69.5 Angstrom and c = 87.2 Angstrom a nd diffract to at least 2.2 Angstrom resolution. A packing density value of 2.55 Angstrom(3)/Da is consistent with one subunit in the asymmetric unit.