HIGH-LEVER PRODUCTION FROM A BACULOVIRUS EXPRESSION SYSTEM AND BIOCHEMICAL-CHARACTERIZATION OF HUMAN GMP SYNTHETASE

GMP synthetase, a key enzyme in the de novo synthesis of guanine nucle otides, is a potential target for immunosuppression and anticancer che motherapy. In order to closely examine the catalytic mechanism and act ive-site topography of this enzyme, large amounts of pure protein are needed. Catalytically active human GMP synthetase was expressed in a b aculovirus system. A high-level production system has been established from which the yield of pure protein is routinely more than 50 mg/10 liters of cell culture. The recombinant enzyme was purified to homogen eity and characterized. Like native GMP synthetase, the recombinant en zyme was resolved into two forms by ion exchange chromatography. The t wo forms are both monomers and they differ in their isoelectric points . There is no evidence that these forms are in equilibrium or intercon vertible. Protein sequence analysis reveals that both forms are blocke d at the amino-terminus and they are essentially identical in sequence . Since they can be produced by a cDNA with a single open reading fram e, we believe that they represent post-translational modification vari ants. The recombinant GMP synthetase is not distinguishable from the n ative enzyme in terms of chromatographic profiles, subunit composition , molecular weight, and kinetic properties. The inhibition constants a nd the modes of inhibition toward decoyinine, a selective inhibitor of GMP synthetase, are also the same as the native enzyme. The high-leve l production of active enzyme is invaluable to the determination of th e three-dimensional structure and the discovery of potent and selectiv e drug candidates. (C) 1995 Academic Press, Inc.