Glutamate Decarboxylase of Escherichia coli:
Expression of gadA Gene, Purification
and Properties of GAD

A. A. Shul’ga, F. T. Kurbanov, R. R. Khristoforov, E. L. Darii, and B. S. Sukhareva

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 117984 Russia;
E-mail: suhareva@genome.eimb.alarn.ru

Received November 13, 1998

Abstract—A method for obtaining recombinant glutamate decarboxylase [EC 4.1.1.15] from E. coli is
described. The enzyme catalyzes L-glutamate conversion to -aminobutyric acid and carbon dioxide. The gadA
gene was cloned on the basis of the known nucleotide sequence using PCR with E. coli TG1 genomic DNA.
Gene expression was carried out in pGEMEX1 vector and E. coli strain BL21(DE3). The enzyme specific activ-
ity reached 4.0–7.3 un./mg wet wt, which is an order of magnitude higher than that for natural wild-type E. coli
600. The method for enzyme isolation was modified, and homogeneous glutamate decarboxylase preparations
of specific activity 171–176 un./mg protein were obtained. The enzyme homogeneity was confirmed by PAGE
and the corresponding OD₂₈₀-to-OD₄₂₀ ratio. The specific activity, k_cat, and absorption spectra of the recombi-
nant protein were identical to those of the natural enzyme isolated from E. coli 600.

Key words: glutamate decarboxylase, E. coli, gadA, PCR, cloning, expression, GAD, purification, catalytic
and spectral properties

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