High-yield expression of properly folded insulinoma-associated protein intracellular domain (IA-2ic) in Escherichia coli.
The intracellular domain of insulinoma-associated protein (IA-2), IA-2ic, is a prominent antigen in autoimmune diabetes, and autoantibodies to it are early markers of the disease. The high-yield expression of properly folded IA-2ic is needed for basic research and crucial for low-cost immunoassays aimed at the detection of these autoantibodies in diagnostic and preventive medicine. In previous work, the expression of IA-2ic fused to glutathione S-transferase or to a biotinylatable peptide was reported; however, these methods had very poor yield. Here we show that, utilizing a codon-optimized gene, up to 80 mg of pure and properly folded autoantigen per litre of Escherichia coli culture may be obtained. Furthermore, the addition of a C-terminal His-tag greatly facilitates IA-2ic purification without compromising either its immunoreactivity or its expression yield. To take advantage of the recombinant antigen, an enzyme immunoassay format was developed which proved to be highly specific and sensitive.
Sica
Mauricio P
MP
School of Pharmacy and Biochemistry, University of Buenos Aires and IDEHU-CONICET, Buenos Aires, Argentina.
Primo
María E
ME
Ermácora
Mario R
MR
Poskus
Edgardo
E
D001324:Autoantigens; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D001324:Autoantigens; D008565:Membrane Proteins; D011994:Recombinant Proteins; C516862:PTPRN protein, human; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8