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

Abstract

Tartrate-resistant acid phosphatase (TRAP) is an enzyme expressed in bone-resorbing osteoclasts and certain tissue macrophages in human tissues. The functions of TRAP in biological systems are not known. Elucidation of the three-dimensional structure of the active site could yield important information about the physiological substrate(s) of the enzyme. We have produced recombinant rat bone TRAP using a baculovirus expression vector system. The production was scaled up to a 30-l bioreactor, and a method of purification in large scale was developed. The enzyme is composed of one 34 kDa polypeptide chain. Trypsin digestion resulted in a preparation where two subunits of ~23 kDa and ~16 kDa appeared after disulfide reduction. Trypsin digestion activated the enzyme. We generated monoclonal antibodies against recombinant TRAP. One of the selected antibodies detected the 23 kDa subunit in Western blotting. The reduced and oxidized forms of the enzyme could be separated by Mono-S cation-exchange chromatography. Crystals of TRAP have been obtained with ammonium sulfate/polyethylene glycol as precipitant. They belong to space group P212121 or P21212 with unit cell dimensions a = 57.2 Å, b = 69.5 Å, and c = 87.2 Å and diffract to at least 2.2 Å resolution. A packing density value of 2.55 Å3/Da is consistent with one subunit in the asymmetric unit.