ABSTRACT
Purity testing of recombinant DNA (rDNA) proteins using slab gel electrophoresis in conjunction with scanning densitometry is time consuming and labor intensive and is difficult to reproduce because the dyes used for visualizing proteins do not bind in a stoichiometric fashion for all proteins. The present report describes a micellar capillary zone electrophoresis (MCZE) procedure that overcomes these difficulties. The MCZE method was evaluated to estimate protein purity of hydrophobic cytomegalovirus proteins, expressed E. coli, and highly glycosylated hepatitis C virus proteins, expressed in Chinese hamster ovary cells. The results obtained by the MCZE procedure correlated very well with the purity results quantitated by the conventional slab gel electrophoresis method using purified Coomassie Brilliant Blue dye to reduce anomalies. MCZE may serve as an alternative method for in-process and purity testing of rDNA proteins.
Subject(s)
Recombinant Proteins/isolation & purification , Animals , CHO Cells , Cloning, Molecular , Cricetinae , Cytomegalovirus , DNA, Ribosomal , Electrophoresis, Capillary/methods , Electrophoresis, Polyacrylamide Gel/methods , Escherichia coli , Glycosylation , Hepacivirus , Molecular Weight , Recombinant Proteins/analysis , Recombinant Proteins/standards , Reproducibility of Results , Sensitivity and Specificity , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Viral Proteins/analysis , Viral Proteins/isolation & purification , Viral Proteins/standardsABSTRACT
Antineutrophil cytoplasmic antibodies (ANCAs) identified in the serum of 50 to 80% of ulcerative colitis (UC) patients yield a perinuclear staining pattern (pANCA) with alcohol-fixed neutrophils. The ANCAs of UC are distinguishable from those described for Wegener's granulomatosis and other vasculitidies. These various non-UC ANCAs recognize neutrophil granule constituents, but the antigenic moiety specific for the UC pANCA remains unknown. Although the perinuclear nature of some ANCA reactions is an artifact of the alcohol fixation of neutrophils, which causes cytoplasmic granules to redistribute around the nucleus, the UC pANCA reaction has been found not to be similarly affected. We postulated a nuclear localization for the UC-associated pANCA antigen and used both confocal laser microscopy and immunoelectron microscopy to examine the neutrophil reaction of UC-associated pANCA-containing sera. Confocal microscopy revealed a nuclear reaction for 88% (22/25) of the sera with 72% (18/25) showing the reaction localizing to the inner side of the nuclear (membrane) periphery. Immunoelectron microscopy showed that the UC-associated pANCA reaction localized primarily over chromatin concentrated toward the nuclear periphery, although the sera did not recognize double-stranded DNA. These results confirm the nuclear localization of the UC-associated pANCA antigen.
