Brain Glutamate Decarboxylase Cloned in λgt-11: Fusion Protein Produces γ-Aminobutyric Acid

• Published in: Science (American Association for the Advancement of Science) Vol. 232; no. 4754; pp. 1138 - 1140
• Main Authors: Kaufman, Daniel L., McGinnis, James F., Krieger, Neil R., Tobin, Allan J.
• Format: Journal Article
• Language: English
• Published: Washington, DC The American Association for the Advancement of Science 30-05-1986; American Association for the Advancement of Science
• Subjects: Amino acids; Animals; Antibodies; Antiserum; Bacteria; Biological and medical sciences; Biotechnology; Brain; Brain - enzymology; Cell physiology; Cloning, Molecular; Complementary DNA; DNA - genetics; Epitopes; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; gamma-Aminobutyric Acid - biosynthesis; Genetic engineering; Genetic screening; Genetic technics; Glutamate Decarboxylase - biosynthesis; Glutamate Decarboxylase - genetics; Glutamate Decarboxylase - metabolism; Humans; Immunology; Libraries; Methods. Procedures. Technologies; Mice; Molecular and cellular biology; Molecular cloning; Neurotransmission; phage lambda; Rats; Recombinant Proteins - biosynthesis; Recombinant Proteins - metabolism; Bactériophage lambda; Enzyme; Brain (Vertebrata); Glutamate decarboxylase; Gene library; Immunological method; Virus; Lambda phage group; Enzymatic activity; Complementary DNA; Styloviridae; Neurotransmitter; Bactériophage; Molecular cloning
• ISSN: 0036-8075; 1095-9203
• DOI: 10.1126/science.3518061

Glutamate decarboxylase (GAD; E.C. 4.1.1.15) converts glutamate to γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the vertebrate central nervous system. This report describes the isolation of a GAD complementary DNA clone by immunological screening of a λgt-11 brain complementary DNA expression library. The fusion protein produced by this clone catalyzes the conversion of glutamate to GABA and carbon dioxide, confirming its identity as GAD. Antibodies to β-galactosidase remove GAD enzymatic activity from solution, showing that this activity is associated with the fusion protein. In immunoblotting experiments all three available antisera to GAD reacted with the fusion polypeptide and with two major polypeptides (molecular size, 60,000 and 66,000 daltons) in brain extracts.