Molecular cloning and expression of the Fabs of human autoantibodies in Escherichia coli. Determination of the heavy or light chain contribution to the anti-DNA/-cardiolipin activity of the Fab
| Autor: | S, Kumar, J, Kalsi, C T, Ravirajan, A, Rahman, D, Athwal, D S, Latchman, D A, Isenberg, L H, Pearl |
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| Rok vydání: | 2000 |
| Předmět: |
Chromatography
Cardiolipins Blotting Western Immunoblotting Enzyme-Linked Immunosorbent Assay DNA Thymus Gland Polymerase Chain Reaction Recombinant Proteins Immunoglobulin Fab Fragments Immunoglobulin G Escherichia coli Animals Humans Cattle Electrophoresis Polyacrylamide Gel Immunoglobulin Light Chains Cloning Molecular Immunoglobulin Heavy Chains Autoantibodies Plasmids |
| Zdroj: | The Journal of biological chemistry. 275(45) |
| ISSN: | 0021-9258 |
| Popis: | The Fabs of three human autoantibodies (B3/33H11, anti-DNA; UK4, anti-phospholipid) and six related hybrids have been cloned, expressed in Escherichia coli, and purified to homogeneity. SDS-polyacrylamide gel electrophoresis and Western blot analysis of the recombinant Fab demonstrated the purified Fab to be of correct size and in assembled form. Protein expression levels of up to 5-9 mg per liter of culture were achievable. A sensitive and reliable comparative anti-DNA enzyme-linked immunosorbent assay, involving a defined biotinylated 35-mer oligonucleotide in its single- or double-stranded form, is also described. Crithidia assay and anti-DNA or anti-cardiolipin antibody enzyme-linked immunosorbent assay analyses demonstrated convincing binding of the recombinant Fab proteins to DNA/cardiolipin, confirming the expression of functional molecule. The comparative DNA/cardiolipin binding analyses of the nine Fabs revealed that the anti-DNA (light, B3/33H11) or anti-cardiolipin (heavy, UK4) activity lies predominantly on one of the two chains. However, a compatible partner chain is necessary for optimum antigen binding activity of the antibody. |
| Databáze: | OpenAIRE |
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