Identification of an inhibitory element within the human 68-kDa (U1) ribonucleoprotein antigen.

Abstract

Various nuclear proteins are the major targets of autoimmune responses in various rheumatic disorders. In particular, autoantibodies directed against a 68-kDa protein associated with the (U1) RNA-containing small nuclear ribonucleoprotein complexes typically occur in sera of patients with mixed connective tissue disease and related rheumatic disorders, such as systemic lupus erythematosus, and therefore are very useful as a serological marker. For establishing powerful immunoassays, it was necessary to generate recombinant human P68 antigen as the antigenic target. In this study we demonstrated that the cDNA coding for the full-length human P68 antigen could not be expressed by a traditional bacterial vector system due to a putative inhibitory sequence designated as inhibitory sequence X which is located between the autoreactive domains C' and D' of the human P68 antigen. The construction of corresponding hybrid plasmids carrying two functional and independent gene blocks indicated the trans-active function of the inhibitory sequence X, which could be localized by expression studies of various deletion constructs. Comparable Northern blot analysis clearly demonstrated that the inhibitory sequence X could act on the translation of the P68 mRNA. After excision of the inhibitory sequence X a dramatic increase in the production of recombinant human P68 antigen was observed.

• Authors: W Northemann, H Berg, G Stahnke, M Walter, N Hunt, S Fenning
• Journal: Protein expression and purification (Protein Expr Purif) Vol. 6 Issue 6 Pg. 748-56 (Dec 1995) ISSN: 1046-5928 [Print] United States
• PMID: 8746626 (Publication Type: Journal Article)

Chemical References

• Autoantibodies
• Autoantigens
• DNA, Complementary
• Recombinant Proteins
• Ribonucleoprotein, U1 Small Nuclear

Topics

• Amino Acid Sequence
• Autoantibodies (analysis)
• Autoantigens (chemistry, genetics)
• Base Sequence
• Chromosome Mapping
• Cloning, Molecular
• DNA, Complementary (genetics)
• Escherichia coli (genetics)
• Gene Expression
• Humans
• Immunoassay
• Mixed Connective Tissue Disease (diagnosis, immunology)
• Molecular Sequence Data
• Molecular Weight
• Plasmids (genetics)
• Recombinant Proteins (chemistry, genetics, immunology)
• Ribonucleoprotein, U1 Small Nuclear (chemistry, genetics, immunology)