Replacement of methionine-161 with threonine eliminates a major by-product of human glutamate decarboxylase 65-kDa variant expression in Escherichia coli.
Most insulin-dependent diabetes mellitus patients gen-erate conformational autoantibodies to the islet-cell 65-kDa variant of human glutamate decarboxylase (GAD65), and several immunochemical tests for the early detection of type-1 diabetes rely on GAD65 antibody (GADA) assessment using properly folded recombinant GAD65 as the antigen. In addition, preventive therapies based on tolerization by GAD65 administration may be available in the near future. Therefore, there exists a strong interest in a facile and economically sound expression procedure for this antigen. Several attempts to produce, in native form, wild-type GAD65 in Escherichia coli have failed. However, this difficulty was recently surmounted in our laboratory by expressing GAD65 as a fusion protein with thioredoxin [Papouchado, Valdez, Ghiringhelli, Poskus and Ermácora (1997) Eur. J. Biochem. 246, 350-359]. In this work, a new GAD65 hybrid gene was prepared by joining engineered cDNA obtained from human and rat tissues. The new gene was modified additionally to finally code for human GAD65 with a single amino-acid substitution: Met-161-->Thr. This change impeded the co-expression of a 48-kDa by-product from an internal translation site. Also, a second 58-kDa by-product was identified as a GAD65 C-terminal proteolytic fragment that co-purifies with thioredoxin-M161T GAD65. The new GAD65 variant was expressed and easily purified, yielding an antigen that performed equally or better than wild-type GAD65 in the reference radiobinding assay for GADA. The procedure provides an inexpensive source of large amounts of fully active and immunochemically competent GAD65.
Santos
J
J
Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Argentina, Roque Sáenz Peña 180, (1876) Bernal, Argentina.
Antón
E A
EA
Buslje
C M
CM
Valdez
S N
SN
Villanueva
A L
AL
Sica
M
M
Iacono
R
R
Maffia
P
P
Poskus
E
E
Ermácora
M R
MR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005968:Glutamate Decarboxylase / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008715:Methionine; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism; D013912:Threonine
D011994:Recombinant Proteins; D013912:Threonine; D013879:Thioredoxins; D008715:Methionine; D005968:Glutamate Decarboxylase