Expression and purification of recombinant mouse fibrillarin.
Fibrillarin is a 34-kDa nucleolar protein associated with many of the small nucleolar ribonucleoprotein (snoRNP) particles and plays a role in ribosomal RNA processing. A subset of patients with the systemic autoimmune disease Scleroderma produce autoantibodies against fibrillarin and it is a genetically restricted target of murine mercury-induced autoimmunity. To aid in characterizing the antigenicity of fibrillarin, we have constructed two forms of mouse fibrillarin. The wild-type clone contains two cysteine residues that enable the protein to form an intramolecular disulfide bond, whereas the mutant clone contains alanine replacements which cannot form the disulfide bond. We have successfully expressed and purified both wild-type and mutant recombinant mouse fibrillarin using nickel-chelation chromatography. The combination of T7 promoter-driven expression vector pET28 and Escherichia coli strain JM109(DE3) induced at 25 degrees C yielded up to 19 mg of 94% pure recombinant protein per liter of culture. As the antigenicity of fibrillarin requires the full-length protein, the purification protocol was optimized for isolation of the full-length protein by the addition of N- and C-terminal T7 Tag and FLAG epitope sequences to the fibrillarin sequence. Anti-peptide antibodies were used in immunoblot to identify conditions favoring minimal proteolysis of recombinant protein. Both wild-type and mutant recombinant fibrillarin, purified under denaturing conditions and in the presence of 2-mercaptoethanol, were recognized by anti-fibrillarin antibodies from Scleroderma patients and exhibited structural similarities to eukaryotic and in vitro translated fibrillarin.
Pearson
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W. M. Keck Autoimmune Disease Center, Department of Molecular and Experimental Medicine, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Reimonenq
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Pollard
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D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002868:Chromosomal Proteins, Non-Histone / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D004274:DNA, Recombinant / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008627:Mercuric Chloride; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D001324:Autoantigens; D002868:Chromosomal Proteins, Non-Histone; D004274:DNA, Recombinant; D004220:Disulfides; D011994:Recombinant Proteins; C047458:fibrillarin; D008627:Mercuric Chloride