A novel method for expression and large-scale production of human brain l-glutamate decarboxylase.
l-Glutamate decarboxylase (GAD; EC 4.1.1.15) is the rate-limiting enzyme involved in the synthesis of gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain. Imbalance in the conversion of glutamate to GABA has been implicated in a host of human diseases. Studies on the structure, function, and therapeutic use of GAD have been precluded by insufficient quantities of purified active enzyme. Here we report a novel methodology for the expression and large-scale production of enzymatically active, pure, recombinant human GAD65 and GAD67. This method circumvents the sequestering of expressed protein into insoluble inclusion bodies and reduces production of truncated proteins. The availability of sufficient quantities of purified HGAD65 and HGAD67 has allowed for the production of specific polyclonal antibodies that discriminate between the two isoforms. This methodology, in addition to providing key human brain enzymes, may be generally applicable to other systems.
Davis
K M
KM
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas.
Foos
T
T
Bates
C S
CS
Tucker
E
E
Hsu
C C
CC
Chen
W
W
Jin
H
H
Tyburski
J B
JB
Schloss
J V
JV
Tobin
A J
AJ
Wu
J Y
JY
D001921:Brain / Q000201:enzymology*; D003001:Cloning, Molecular; D004926:Escherichia coli; D005968:Glutamate Decarboxylase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013574:Synaptosomes / Q000201:enzymology
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; C401141:glutamate decarboxylase 2