Expression in Saccharomyces cerevisiae of antigenically and enzymatically active recombinant glutamic acid decarboxylase.
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.
Law
R H
RH
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic, Australia.
Rowley
M J
MJ
Mackay
I R
IR
Corner
B
B
D003922:Diabetes Mellitus, Type 1 / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005968:Glutamate Decarboxylase / Q000235:genetics*; D006801:Humans; D007163:Immunosorbent Techniques; D011994:Recombinant Proteins / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D016622:Silver Staining
D011994:Recombinant Proteins; D005968:Glutamate Decarboxylase