Expression of recombinant proteinase 3, the autoantigen in Wegener's granulomatosis, in insect cells.
Proteinase 3 (PR3) is the major autoantigen for anti-neutrophil cytoplasmic antibodies (ANCA) in patients with Wegener's granulomatosis. Little is known about the major antigenic sites on PR3. To facilitate epitope mapping, PR3 was cloned in insect cells using a baculovirus expression system. Four different sequences of the PR3 cDNA were amplified by PCR: two clones containing the pro-peptide of PR3 with or without a His-tag (rproPR3-his and rproPR3, respectively) and two clones without the pro-peptide and with or without a His-tag (rPR3-his and rPR3, respectively). The PR3 sequences were cloned behind the polyhedrin promoter and the honeybee melittin signal peptide enabling secretion of rPR3. Plasmids were transposed into the genome of baculovirus, and wild types as well as PR3-containing virus genomes were transfected into Sf21 insect cells. All four rPR3 variants were secreted into the medium and were recognized by anti-neutrophil PR3 rabbit serum and by at least two anti-PR3 monoclonal antibodies. Mature forms of PR3 were recognized by almost all patient sera, whereas the pro-forms of PR3 were recognized by 14 of 18 PR3-ANCA sera tested. On SDS-PAGE, the four rPR3 forms migrated at approximately 32 kDa. RPR3-his and rproPR3-his could be purified by means of this His-tag. In conclusion, especially the mature rPR3s are well recognized by PR3-ANCA sera. The presence of a C-terminal His-tag facilitated purification of His-tagged rPR3. Thus, rPR3 expressed in insect cells can be used as a tool for diagnostic tests as well as for epitope mapping studies.
Department of Internal Medicine, University Hospital Groningen, Groningen, The Netherlands.
Y M
YM
Van der Geld
M L F
ML
Smook
M G
MG
Huitema
M C
MC
Harmsen
P C
PC
Limburg
C G M
CG
Kallenberg
D000818:Animals; D001324:Autoantigens / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D016367:Baculoviridae / Q000235:genetics; D001666:Binding Sites, Antibody / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D002999:Clone Cells; D003001:Cloning, Molecular / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D014890:Granulomatosis with Polyangiitis / Q000276:immunology*; D006639:Histidine / Q000235:genetics; D006801:Humans; D007106:Immune Sera / Q000378:metabolism; D053146:Myeloblastin; D010446:Peptide Fragments / Q000032:analysis; D011498:Protein Precursors / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D018411:Spodoptera / Q000235:genetics* / Q000821:virology; D013997:Time Factors
D001324:Autoantigens; D007106:Immune Sera; D010446:Peptide Fragments; D011498:Protein Precursors; D011994:Recombinant Proteins; D006639:Histidine; D012697:Serine Endopeptidases; D053146:Myeloblastin