Expression and inhibition studies of recombinant human brain 4-aminobutyrate aminotransferase

Author

Li Weikai

Date of Award

8-1997

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Jorge E. Churchich

Committee Members

Cynthia Peterson, Ranjan Ganguly

Abstract

The gene for human brain 4-aminobutyrate aminotransferase (hGABAT) cloned in either the PET 12a vector or the PET30a vector, and the pT-Trx plasmid that contains the genes coding for thioredoxin were co-transformed into the E. coli strain BL21 (DE3). The recombinant hGABAT was then coexpressed with thioredoxin by induction of IPTG. Active enzyme was detected by both a fluorometric assay and a spectrophotometric assay. The production of soluble, active recombinant hGABAT was in contrast to the formation of insoluble, inactive aggregates when the hGABAT was expressed alone in BL21 (DE3).

The hGABAT expressed from the pET12a vector was purified to over 90% by chromatography on a DE52 cellulose column and a hydroxyapatite column. A hGABAT fusion protein bearing an N-terminal His-tag and S-tag (pET30a-hGABAT) was purified using a His*Bind Ni-column. Part of the sample was subjected to further digestion with thrombin to give the recombinant hGABAT without a His-tag. The expression of all recombinant enzyme species was confirmed 1) by enzyme activity, measured as specific transamination reaction between β-alanine and α-ketoglutarate, and 2) by immunochemical methods using a mouse monoclonal antibody against pig brain GAB AT. The recombinant fusion protein of GABAT with the N-terminal His-tag and S-tag was also detected with a S-protein alkaline phosphatase conjugate.

Kinetic constants, K_m and V_max were evaluated for the pET12a-hGABAT using the max’ two substrates, GABA and α-ketoglutarate. The pET12a-hGABAT, pET30a-hGABAT, and thrombin digested pET30a-hGABAT were evaluated for inhibition by vigabatrin and acetylenic GABA. Vigabatrin was found to have a weak effect on inactivation of recombinant hGABAT species. The inhibition behavior of the three hGABAT species was compared with that of pig brain GAB AT; the inhibitors, vigabatrin (racemic mixture), acetylenic GABA S (+) isomer and R(-) isomer, and hydoxyamine were compared for their inhibition effect. The inhibition of pET12a-hGABAT by vigabatrin and acetylenic GABA S (+) isomer were subjected to kinetic analysis, and the K_i and k₂ were obtained for each inhibitor.

Recommended Citation

Weikai, Li, "Expression and inhibition studies of recombinant human brain 4-aminobutyrate aminotransferase. " Master's Thesis, University of Tennessee, 1997.
https://trace.tennessee.edu/utk_gradthes/10588