Cost-effective production of "GABase" for spectrophotometric determination of γ-aminobutyrate (GABA) levels or glutamate decarboxylase activity.

γ-aminobutyrate (GABA) is a non-proteinogenic amino acid produced by glutamate decarboxylase (GAD) that functions as a vital neurotransmitter in animals, and as an important metabolite and signaling molecule in plants and microbes. "GABase" consists of a mixture of recombinant GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSDH) that is widely used for spectrophotometric quantification of glutamate decarboxylase (GAD) activity or GABA levels in tissue extracts. Both can be conveniently monitored at 340 nm owing to the sequential conversion of GABA into succinate by GABA-T and SSDH, and concomitant reduction of NADP into NADPH by SSDH. Currently, these assays rely on commercially available GABase from . However, the excessive cost of commercial GABase prompted us to develop an inexpensive and rapid "DIY" method for producing GABase by cloning, expressing and purifying His-tagged GABA-T and SSDH from . We validated our in-house GABase preparation by comparing GAD activities and GABA levels of the model plant with those obtained using commercial GABase. Both plasmids for expressing His-GABA-T and His-SSDH have been deposited into AddGene (www.addgene.com). Our protocols for producing and using recombinant GABase should be of interest to any researcher who studies eukaryotic or prokaryotic GABA and/or GAD activity.