Isolation and Comparison of Natural and Recombinant Human CENP-A Autoantigen

Anticentromere antibodies (ACA) are associated with systemic sclerosis (scleroderma) patients exhibiting the more benign or so called limited manifestation of the disease (lSSc). ACA reactivity is directed against multiple polypeptide targets, the smallest of which is designated CENP-A. CENP-A is no...

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Bibliographic Details
Published in:Journal of autoimmunity 1998-12, Vol.11 (6), p.611-619
Main Authors: Martinez, Antigona, Sun, Dongxu, Billings, Peter B, Swiderek, Kristine M, Sullivan, Kevin F, Hoch, Sallie O
Format: Article
Language:English
Subjects:
Animals
anticentromere antibodies (ACA)
Autoantigens - biosynthesis
Autoantigens - immunology
Autoantigens - isolation & purification
Baculoviridae - genetics
Biological and medical sciences
Centromere Protein A
Chromatography, Agarose
Chromatography, High Pressure Liquid
Chromosomal Proteins, Non-Histone - biosynthesis
Chromosomal Proteins, Non-Histone - immunology
Chromosomal Proteins, Non-Histone - isolation & purification
Electrophoresis, Polyacrylamide Gel
General aspects
HeLa Cells
human CENP-A
Humans
Immunopathology
Medical sciences
recombinant CENP-A
Recombinant Proteins - biosynthesis
Recombinant Proteins - immunology
Recombinant Proteins - isolation & purification
scleroderma
Sodium Dodecyl Sulfate
Spodoptera - metabolism
Spodoptera - virology
SSc
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Description
Summary:Anticentromere antibodies (ACA) are associated with systemic sclerosis (scleroderma) patients exhibiting the more benign or so called limited manifestation of the disease (lSSc). ACA reactivity is directed against multiple polypeptide targets, the smallest of which is designated CENP-A. CENP-A is not an abundant cellular constituent; therefore to maximize recovery, we developed a protocol with a minimum of steps to isolate CENP-A from a human cell line. The trace cellular amount of this protein clearly dictated the production of its recombinant counterpart to facilitate determination of the role of the CENP-A antigen in scleroderma pathogenesis. Here we describe the eukaryotic expression of CENP-A cDNA using baculovirus-mediated infection of insect cells. The non-fusion recombinant protein spans the natural residues of the human CENP-A protein and rCENP-A followed the same chromotographic sequence for purification as did the natural source. The availability of thebona fideantigen provided a critical standard upon which to document authenticity of the recombinant polypeptide. The two forms of this antigen have been compared and shown to exhibit similar physical and antigenic properties.
ISSN:0896-8411
1095-9157
DOI:10.1006/jaut.1998.0249