An efficient method for enrichment of histidyl-tRNA synthetase (Jo-1 antigen) from HeLa cells.
A rapid method has been developed for enrichment of Jo-1 antigen (histidyl-tRNA synthetase) from HeLa cells. The enzyme has been prepared from post-ribosomal supernatant by successive chromatography with Blue Sepharose and Poly-U-Sepharose, followed by DEAE-high performance liquid chromatography (HPLC). By this method, enzyme could be obtained within 4 days of HeLa cell harvesting, with 40% recovery of the enzymatic activity. The apparent native molecular size of the enzyme as determined by HPLC-size exclusion column chromatography was approximately 120 kDa. Under denaturing conditions using SDS-polyacrylamide gel electrophoresis the enzyme subunit size was approximately 55 kDa. The antigen preparation, although not homogeneous, was found to react only with anti-Jo-1 positive antisera when tested by immunoblotting with many patient sera of defined autoantibody specificities, making the preparation useful for immunologic studies of anti-Jo-1 antibodies.
D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification*; D000974:Antibodies, Antinuclear / Q000032:analysis; D001324:Autoantigens / Q000302:isolation & purification*; D001327:Autoimmune Diseases / Q000097:blood; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006367:HeLa Cells / Q000201:enzymology / Q000276:immunology*; D006643:Histidine-tRNA Ligase / Q000302:isolation & purification*; D006801:Humans; D008970:Molecular Weight; D012216:Rheumatic Diseases / Q000097:blood / Q000276:immunology; D012685:Sepharose / Q000031:analogs & derivatives / Q000494:pharmacology
D000974:Antibodies, Antinuclear; D001324:Autoantigens; C046750:Jo-1 antibody; C030188:poly U-sepharose; C029483:Blue-Sepharose; D003101:Collodion; D012685:Sepharose; D000604:Amino Acyl-tRNA Synthetases; D006643:Histidine-tRNA Ligase