High-level 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 nucleotides, is a potential target for immunosuppression and anticancer chemotherapy. In order to closely examine the catalytic mechanism and active-site topography of this enzyme, large amounts of pure protein are needed. Catalytically active human GMP synthetase was expressed in a baculovirus 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 homogeneity and characterized. Like native GMP synthetase, the recombinant enzyme was resolved into two forms by ion-exchange chromatography. The two forms are both monomers and they differ in their isoelectric points. There is no evidence that these forms are in equilibrium or interconvertible. Protein sequence analysis reveals that both forms are blocked at the amino-terminus and they are essentially identical in sequence. Since they can be produced by a cDNA with a single open reading frame, we believe that they represent post-translational modification variants. The recombinant GMP synthetase is not distinguishable from the native enzyme in terms of chromatographic profiles, subunit composition, molecular weight, and kinetic properties. The inhibition constants and the modes of inhibition toward decoyinine, a selective inhibitor of GMP synthetase, are also the same as the native enzyme. The high-level production of active enzyme is invaluable to the determination of the three-dimensional structure and the discovery of potent and selective drug candidates.
Institute of Biochemistry and Cell Biology, Syntex Discovery Research, Palo Alto, California 94304, USA.
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L
Lou
J
J
Nakamura
S
S
Tsing
B
B
Nguyen
J
J
Chow
K
K
Straub
H
H
Chan
J
J
Barnett
D000818:Animals; D019731:Carbon-Nitrogen Ligases; D002460:Cell Line; D002845:Chromatography; D015870:Gene Expression; D006801:Humans; D007526:Isoelectric Point; D007700:Kinetics; D008025:Ligases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D008970:Molecular Weight; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D018411:Spodoptera
D011994:Recombinant Proteins; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C020828:GMP synthase (glutamine-hydrolyzing)