Expression of the Fabs of human auto-antibodies in Escherichia coli: optimization and determination of their fine binding characteristics and cross-reactivity.
The Fabs of three human auto-antibodies (B3/33H11, anti-DNA; UK4, anti-phospholipid) and six related hybrids have been cloned and expressed in Escherichia coli, and their relative binding to single-stranded or double-stranded DNA or to cardiolipin has been assessed in the presence of modulators (salts and serum). We describe optimized conditions that have led to significant improvement in the quality and quantity of the purified auto-antibodies. Protein expression of the assembled and functionally active Fabs was achievable with a yield of up to 5 to 9 mg/l of culture. The comparative DNA/cardiolipin-binding analyses of the nine Fabs in the presence of modulators demonstrated that B3 and 33H11 L chains possess both anti-DNA and anti-cardiolipin activities. This is the first report of the demonstration that both anti-DNA and anti-cardiolipin activities may lie on the same light chain of a human auto-antibody. We provide evidence that the auto-antibodies that appeared to be similar, in that they bound DNA or cardiolipin in conventional ELISA immunoassays, exhibited significant difference in their cross-reactivity and binding to the antigen in the presence of modulators. Such auto- antigen specificity and/or cross-reactivity may dictate the potential of an auto-antibody to cause pathogenicity and may provide an explanation as to why apparently similar auto-antibodies behave differently in vivo.
Kumar
S
S
Centre for Rheumatology, and Department of Biochemistry and Molecular Biology, University College London, UK. sanjeev@icr.ac.uk
Kalsi
J
J
Latchman
D S
DS
Pearl
L H
LH
Isenberg
D A
DA
D000818:Animals; D000974:Antibodies, Antinuclear / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity / Q000187:drug effects / Q000276:immunology*; D001323:Autoantibodies / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D001324:Autoantigens / Q000276:immunology; D001798:Blood Proteins / Q000494:pharmacology; D015153:Blotting, Western; D002308:Cardiolipins / Q000276:immunology; D002417:Cattle; D003429:Cross Reactions / Q000187:drug effects / Q000276:immunology*; D004247:DNA / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D007146:Immunoglobulin lambda-Chains / Q000276:immunology; D008180:Lupus Erythematosus, Systemic / Q000276:immunology / Q000503:physiopathology; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000276:immunology; D010710:Phosphates / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D017411:Ribonucleoproteins, Small Nuclear; D012965:Sodium Chloride / Q000494:pharmacology; D055517:snRNP Core Proteins
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; D001798:Blood Proteins; D002308:Cardiolipins; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007146:Immunoglobulin lambda-Chains; D010446:Peptide Fragments; D010710:Phosphates; D011994:Recombinant Proteins; D017411:Ribonucleoproteins, Small Nuclear; D055517:snRNP Core Proteins; D012965:Sodium Chloride; D004247:DNA