A novel method for expression and large-scale production of human brain L-glutamate decarboxylase

L-Glutamate decarboxylase (GAD; EC 4.1.1.15) is the rate-limiting enzyme in volved in the synthesis of gamma-aminobutyric acid (GABA), the major inhibi tory neurotransmitter in the mammalian brain, Imbalance in the conversion o f glutamate to GABA has been implicated in a host of human diseases, Studie s on the structure, function, and therapeutic use of GAD have been preclude d by insufficient quantities of purified active enzyme. Here we report a no vel methodology for the expression and large-scale production of enzymatica lly active, pure, recombinant human GAD65 and GAD67. This method circumvent s the sequestering of expressed protein into insoluble inclusion bodies and reduces production of truncated proteins. The availability of sufficient q uantities of purified HGAD65 and HGAD67 has allowed for the production of s pecific polyclonal antibodies that discriminate between the two isoforms. T his methodology, in addition to providing key human brain enzymes, may be g enerally applicable to other systems. (C) 2000 Academic Press.