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

• Main Authors: Mingu Gordon Park, Ah-reum Han, Su Yeon Kim, Tai Young Kim, Ho Min Kim, C. Justin Lee
• Format: Article
• Language: English
• Published: Taylor & Francis Group 2021-01-01
• Series: Journal of Enzyme Inhibition and Medicinal Chemistry
• Subjects: 4-aminobutyrate transaminase; isolation and purification; high-throughput screening assays; gabaculine; vigabatrin
• Online Access: http://dx.doi.org/10.1080/14756366.2021.1975697

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.