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

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.

DMSO-tolerant ornithine decarboxylase (ODC) tandem assay optimised for high-throughput screening

“…To produce recombinant human ODC with appropriate folding and PTMs in large quantities for HTS, we attempted to utilise a protocol for overexpression of C-terminal 6xHis-tagged human ODC in Expi293F TM cells, followed by Ni-NTA affinity chromatography and size-exclusion chromatography as previously described 36 . Unexpectedly, we were not able to obtain any fraction containing biologically active human ODC ( Figure 2A , bottom panel).…”

“…Then, enhancer was added immediately. After 24 h, the incubation temperature was lowered from 37 °C to 32 °C to slow cell growth rate while further enhancing protein expression yield 36 , 38 . Overexpression of recombinant human ODC1 was monitored for 24 to 48 h via IRES-driven GFP expression in Expi293 TM cells until transfection efficiency was reached to more than 60%.…”

Genome-wide association study of John Henryism in the CARDIA Cohort suggests a molecular mechanism behind lower nicotine addiction rates

“…Transversions (Tv) are typically found to interrupt TF binding [24], and in our case the mutation found in individuals with high JH results in increased GC content. Therefore, one potential pathway to increased smoking rates and nicotine addiction in individuals with low JH is that the ABAT Tv we found results in increased levels of ABAT protein [25][26][27], which catabolizes the neurotransmitter GABA [28], which in turn means a decrease in available GABA for binding to the GABA B receptor resulting in an increase in the nicotine reward [29]. Taken together, individuals with high JH appear to experience less reward from nicotine use and would subsequently be not as susceptible to addiction.…”

Multi-tissue metabolomic profiling reveals potential mechanisms of cocoon yield in silkworms (Bombyx mori) fed formula feed versus mulberry leaves

“…The compound 3-amino-2,3-dihydrobenzoic acid, in the category of amino acids, peptides and analogues, showed higher concentrations in all experimental tissues of the FF silkworms. This compound, also known as gabaculin or 5-aminocyclohexa-1,3-diene-1-carboxylic acid, exists in all living organisms and inhibits the activity of γ-aminobutanoic acid-transaminase ( Park et al, 2021 ) and ornithine aminotransferase ( Li et al, 2016 ). Inhibition of ornithine aminotransferase results in restriction of ornithine synthesis with glutamate (KEGG Module: M00028) as a substrate.…”