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High-yield synthesis and purification of recombinant human GABA transaminase for high-throughput screening assays.
- Source :
- Journal of Enzyme Inhibition & Medicinal Chemistry; Dec2021, Vol. 36 Issue 1, p2016-2024, 9p
- Publication Year :
- 2021
-
Abstract
- Many studies have focussed on modulating the activity of γ-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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 14756366
- Volume :
- 36
- Issue :
- 1
- Database :
- Complementary Index
- Journal :
- Journal of Enzyme Inhibition & Medicinal Chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 154690927
- Full Text :
- https://doi.org/10.1080/14756366.2021.1975697