Isolation and comparison of natural and recombinant human CENP-A autoantigen

Anticentromere antibodies (ACA) are associated with systemic sclerosis (scl eroderma) patients exhibiting the more benign or so called limited manifest ation of the disease (lSSc). ACA reactivity is directed against multiple po lypeptide targets, the smallest of which is designated CENP-A. CENP-A is no t an abundant cellular constituent; therefore to maximize recovery, we deve loped a protocol with a minimum of steps to isolate CENP-A from a human cel l line. The trace cellular amount of this protein clearly dictated the prod uction of its recombinant counterpart to facilitate determination of the ro le of the CENP-A antigen in scleroderma pathogenesis. Here we describe the eukaryotic expression of CENP-A cDNA using baculovirus-mediated infection o f insect cells. The non-fusion recombinant protein spans the natural residu es of the human CENP-A protein and rCENP-A followed the same chromotographi c sequence for purification as did the natural source. The availability of the bona fide antigen provided a critical standard upon which to document a uthenticity of the recombinant polypeptide. The two forms of this antigen h ave been compared and shown to exhibit similar physical and antigenic prope rties. (C) 1998 Academic Press.