Expression of Properly Folded Human Glutamate Decarboxylase 65 as a Fusion Protein in Escherichia Coli

Abstract

Autoantibodies to the islet-cell 65-kDa variant of glutamate decarboxylase (GAD65) are found in most insulin-dependent diabetes mellitus (IDDM) patients many years before the appearance of clinical symptoms of the disease. As IDDM-preventive therapies may be available in the future, an international effort is taking place to develop widely applicable anti-GAD immunochemical tests. These tests would help to detect individuals at risk before the full installation of the disease and to enroll them in prevention programs. Autoantibodies to GAD65 are mostly directed to conformational epitopes, and the enzyme is a complex molecule with a prosthetic group and 15 cysteine residues. Thus, the conformational integrity of GAD65 is essential for an appropriate anti-GAD assay. Isolation of large amounts of GAD65 from pancreas or other tissues is impractical, and no successful production of properly folded GAD65 has been reported in bacteria. Native recombinant GAD65 for immunochemical tests is usually obtained from eukaryotic expression systems. Since the large-scale production of a recombinant protein in an eukaryotic system is expensive and technically difficult, we investigated the expression of GAD65 in Escherichia coli as an alternative. A number of DNA constructs intended to export the enzyme to the periplasmic space or to improve its cytoplasmic solubility were designed and tested. Our results provide a solution to the two main problems associated with the expression of GAD65 in E. coli: misfolding, leading to the formation of inclusion bodies ; and the presence of alternative initiation sites for translation that causes the preferential production of truncated variants of GAD65. We describe here the production of properly folded, fully active, and immunochemically competent GAD65 as an N-terminal fusion protein with thioredoxin. An account of the reactivity of the produced protein with sera of six IDDM patients is also presented.

Expression and characterization of human proinsulin fused to thioredoxin in Escherichia coli

“…Our approach involved the production of soluble cytoplasmic PI by means of the apparently general solubilizing effect of the thioredoxin moiety (LaVallie et al 1993). Although the exact mechanism by which this happens is not known, thioredoxin has been shown to facilitate the soluble expression of several cytokines, peptides and other proteins (Chopra et al 1994;Wilkinson et al 1995;Papouchado et al 1997). In this work, Trx-PI was successfully expressed in the intracellular soluble fraction of E. coli GI724 at 37°C.…”

“…Further significant improvements in the production of the fusion protein could be obtained by optimisation of the bacterial growth conditions. Our findings provide a new example of a protein that is successfully expressed as a thioredoxin fusion protein presenting the immunochemical behaviour of the native protein (Georgiou and Valax 1996;Papouchado et al 1997).…”

Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application

“…High purity levels were achieved, even higher than those reported in the results section (72–77 %), which does not include the contribution of TrxIA-2 ic dimer. Furthermore, these unique properties of the fusion partner allowed us to establish a general platform for expression and purification of soluble Trx fusion proteins, such as TrxGAD and TrxPI [68, 70], along with TrxIA-2 ic , applicable to the laboratory diagnosis of autoimmune DM. The amount of protein obtained with this methodology was suitable for the development of solid phase IA-2A immunoassays; in fact, the result of a standard purification protocol may allow passive adsorption of more than 500 96-well polystyrene microplates.…”

Flow cytometric microsphere-based immunoassay as a novel non-radiometric method for the detection of glutamic acid decarboxylase autoantibodies in type 1 diabetes mellitus

“…The fusion protein TrxGAD65 was expressed in E. coli as previously described 14 . Briefly, E. coli GI698 was transformed with pGAD65 (Trx).…”

“…These approaches are feasible when a handy source of glutamic acid decarboxylase 65 kDa isoform (GAD65) is available for immobilization to microplates (i.e, recombinant protein obtained from modified prokaryotic cells). In this sense, we have previously described the expression of human GAD65 in Escherichia coli as a soluble and properly folded fusion protein with thioredoxin (TrxGAD65) 14 . This method has allowed us to determine the presence of GADA by ELISA 12 , with a similar format to that of Palomer et al 15 .…”