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 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 the bona fide antigen 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.
Martinez
A
A
The Agouron Institute, La Jolla, CA, 92037, USA.
Sun
D
D
Billings
P B
PB
Swiderek
K M
KM
Sullivan
K F
KF
Hoch
S O
SO
D000818:Animals; D001324:Autoantigens / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification*; D016367:Baculoviridae / Q000235:genetics; D000076247:Centromere Protein A; D002847:Chromatography, Agarose; D002851:Chromatography, High Pressure Liquid; D002868:Chromosomal Proteins, Non-Histone / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate; D018411:Spodoptera / Q000378:metabolism / Q000821:virology
D001324:Autoantigens; C000619700:CENPA protein, human; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate