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

Journal of Enzyme Inhibition and Medicinal Chemistry

Ah-reum Han¹  Ho Min Kim²  Tai Young Kim³  Su Yeon Kim³  C. Justin Lee⁴  Mingu Gordon Park⁴ 

[1] Center for Biomolecular and Cellular Structure, Institute for Basic Science (IBS), Daejeon, South Kore;Center for Biomolecular and Cellular Structure, Institute for Basic Science (IBS), Daejeon, South Kore;Graduate School of Medical Science & Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, South Kore;Center for Cognition and Sociality, Institute for Basic Science (IBS), Daejeon, South Kore;KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul, South Kore;Center for Cognition and Sociality, Institute for Basic Science (IBS), Daejeon, South Kore;

关键词: 4-Aminobutyrate transaminase;    isolation and purification;    high-throughput screening assays;    gabaculine;    vigabatrin;   

DOI  :  10.1080/14756366.2021.1975697

来源: Taylor & Francis

【 摘 要 】

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.

【 授权许可】

CC BY