Expression in yeast of antigenically active, recombinant hybrid

Details Expression in yeast of antigenically active, recombinant hybrid Expression in yeast of antigenically active, recombinant hybrid

1999-09-13

2000-12-26

Achutamurthy, Ponnathapu

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

435232, 4353201, 4352523, 435419, 435325, 4352542, 43525421, 43525423, 530350, 536 231, 536 232, 424 945, G01N 3353

Patent

active

061657387

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

This invention relates to the production of an antigenically active hybrid glutamic acid decarboxylase (GAD) molecule as a recombinant protein by expression in eukaryotic host cells, particularly yeasts, and to the use of this recombinant hybrid GAD in the diagnosis and presymptomatic detection of insulin-dependent (Type 1) diabetes mellitus (IDDM). The invention also relates to the use of this hybrid GAD molecule to inhibit or prevent the occurrence of IDDM in individuals who are diagnosed or detected as having presymptomatic IDDM.


BACKGROUND OF THE INVENTION

Insulin dependent diabetes mellitus (DDM) is an autoimmune disease in which there is a characteristic immunological reactivity to a limited set of tissue-specific cytoplasmic autoantigens of pancreatic islet beta cells. Reactivity to one of these autoantigens, glutamic acid decarboxylase (GAD; EC 4.1.1.15), is virtually unique to the disease, the rare exceptions being the neurological disorder, Stiff man syndrome (Solimena et al., 1990; Baekkeskov et al., 1990), and the polyendocrine syndrome Types 1 and 2. Glutamic acid decarboxylase catalyses the conversion of L-glutamic acid to .gamma.-aminobutyric acid (GABA) and carbon dioxide (Erlander et al., 1991). GABA is a major inhibitory neurotransmitter, and hence most research on GAD until recently had concentrated on the role of this enzyme in neural functioning. A new direction developed with the recognition that antibodies to GAD are prevalent in IDDM (Baekkeskov et al., 1990).
GAD exists as 2 isoforms denoted by their calculated molecular weights as GAD65 and GAD67. These differ according to their subcellular location (Erdo and Wolff, 1990; Faulkner-Jones et al., 1993), chromosomal location (Erlander et al. 1991; Karlsen et al., 1991; Bu et al., 1992; Michelsen et al., 1991), amino acid sequence (Bu et al., 1992) and cofactor interactions (Erlander and Tobin, 1991) but have close homology, in man 65% identity and 80% similarity. The greatest divergence between the isoforms occurs in the first 100 amino acids (Bu et al. 1992). The availability of cDNA clones encoding the two GAD isoforms has allowed the expression of these in various systems, including bacteria (Kaufman et al., 1992), Sf9 insect cells using the baculovirus vector (Seissler et al., 1993; Mauch et al., 1993), COS7 monkey cells (Velloso et al., 1993), baby hamster kidney cells (Hagopian et al., 1993), yeast (Powell et al, 1995) and by in vitro translation using rabbit reticulocyte lysate (RRL) (Petersen et al., 1994; Ujihara et al., 1994; Grubin et al., 1994). In addition, a modified GAD65 without the hydrophobic amino acids 245 inclusive of the N-terminal region has been expressed in yeast (Powell, et al., 1996).
The identification of GAD as a major autoantigen of IDDM has led to the extensive use of this antigen in immunoassays for the accurate diagnosis and prediction of IDDM in at-risk populations. Such studies have shown that antibodies to GAD are detectable in patients up to 10 years before the early onset of clinical symptoms (Baekkeskov et al., 1987, Atkinson et al., 1990; Rowley et al., 1992; Chen et al., 1993; Tuomilehto et al., 1994; Myers et al., 1996). These assays have employed autoantigenic GAD derived from two major sources. One source is animal materials, most commonly porcine brain, purified by affinity chromatography, and labelled with radioactive iodine. The other source is in vitro transcription and translation of the cloned human GAD65 gene, using rabbit reticulocyte lysate (RRL) which produces biosynthetically labelled GAD suitable for radioimmunoprecipitation (RIP) assays (Guazzaroti et al., 1995). Expression from RRL has been widely used in diagnostic assays for anti-GAD in human sera but the in vitro expression system has limitations in that only very small amounts, in the order of picomoles, of GAD are produced and the process is very costly. Bacterial expression does not appear to yield GAD that is amenable to use in diagnostic assays, and yields from mammalian cells are unsuitably

REFERENCES:
patent: 5821334 (1998-10-01), Powers
K. Daw et al., "Diabetes 44", Two Distinct Glutamic Acid Decarboxylase Auto-Antibody Specificities in IDDM Target Different Epitopes, pp. 216-220, (1995).
K. Daw et al., "J. Immunol. 156(2)", Glutamic Acid Decarboxylase Autoantibodies in Stiff-Man Syndrome and Insulin-Dependent Diabetes Mellitus Exhibit Similarities in Epitope Recognition, pp. 818-825, (1996).
B. Ziegler et al., "Acta Diabetol 33", Murine Monoclonal Glutamic Acid Decarboxylase (GAD) 65 Antibodies Recognise Autoimmune-Associated GAD Epitope Regions Targeted in Patients with Type 1 Diabetes Mellitus and Stiff-Man Syndrome, pp. 225-231, (1996).
Bu et al. Two human glutamate decarboxylases, 65-kDa GAD and 67-kDa GAD, ar encoded by a single gene. PNAS (1992) 89:2115-2119, Mar. 1992.
GenBank Accession No. NP.sub.- 000808, Mar. 1992.
GenBank Accession No. Q05329, Mar. 1992.

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