High-yield synthesis and purification of recombinant human GABA transaminase for high-throughput screening assays

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Many studies have focussed on modulating the activity of gamma-aminobutyric acid transaminase (GABA-T), a GABA-catabolizing enzyme, for treating neurological diseases, such as epilepsy and drug addiction. Nevertheless, human GABA-T synthesis and purification have not been established. Thus, biochemical and drug design studies on GABA-T have been performed by using porcine GABA-T mostly and even bacterial GABA-T. Here we report an optimised protocol for overexpression of 6xHis-tagged human GABA-T in human cells followed by a two-step protein purification. Then, we established an optimised human GABA-T (0.5 U/mg) activity assay. Finally, we compared the difference between human and bacterial GABA-T in sensitivity to two irreversible GABA-T inhibitors, gabaculine and vigabatrin. Human GABA-T in homodimeric form showed 70-fold higher sensitivity to vigabatrin than bacterial GABA-T in multimeric form, indicating the importance of using human GABA-T. In summary, our newly developed protocol can be an important first step in developing more effective human GABA-T modulators.
Publisher
TAYLOR & FRANCIS LTD
Issue Date
2021-12
Language
English
Article Type
Article
Citation

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, v.36, no.1, pp.2016 - 2024

ISSN
1475-6366
DOI
10.1080/14756366.2021.1975697
URI
http://hdl.handle.net/10203/287848
Appears in Collection
MSE-Journal Papers(저널논문)
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