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Published on: February 25, 2010
Expression in Saccharomyces cerevisiae of antigenically and enzymatically active recombinant glutamic acid
R H Law1, M J Rowley, I R Mackay
1Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic, Australia.
Abstract:
Glutamic acid decarboxylase (GAD) is one of the major autoantigens found in insulin-dependent (Type 1) diabetes mellitus (IDDM). A novel hybrid form of GAD was created by fusing amino acids 1-101 of the human GAD67 protein to amino acids 96-585 of the human GAD65 protein. This hybrid GAD67/65 was expressed constitutively under the control of the phosphoglycerate kinase promoter (PGK1) in the yeast Saccharomyces cerevisiae. Enzymatically active GAD was prepared from yeast lysates by a one-step purification on an affinity column using GAD-1 antibody. The purified hybrid GAD67/65 was radiolabelled with iodine-125 and tested in an immunoprecipitation assay with IDDM sera. Results obtained using the recombinant yeast hybrid GAD67/65 were very similar to those obtained using 125I-labelled porcine GAD. Recombinant yeast hybrid GAD67/65 should have utility for diagnosis and presymptomatic detection of IDDM.
