On the interaction between single chain Fv antibodies and bacterial immunoglobulin-binding proteins.
Using four bacterial immunoglobulin-binding proteins, we have analyzed the binding characteristics of a panel of 34 human single chain Fv antibodies, expressed in E. coli and with known specificity and sequence. Several of the single chain Fv antibodies showed affinity for staphylococcal protein A and peptostreptococcal protein L, but not for the streptococcal proteins G or H. The affinity of the binding was higher for protein L (4.5 and 1.4 x 10(9) M-1) than for protein A (7.7 and 6.7 x 10(8) M-1), using the two single chain Fv antibodies displaying the strongest binding activity to these ligands. The binding was shown to be specific by Western blotting, and the single chain Fv antibodies could be purified from crude bacterial culture media by affinity chromatography on protein L- or A-Sepharose. Protein A, which has affinity for the VH domain of the scFv antibodies, was tested against scFv antibodies containing VH1, VH3, VH4 and VH5 domains, and its binding was restricted to approximately half of the scFv antibodies with a VH3 domain. Protein L, which has affinity for the VL domain, was tested against kappa 1, kappa 4, lambda 1, lambda 2 and lambda 3 domains, and it bound all kappa 1 domains, one lambda 2 and one lambda 3 domain. Comparison of the amino acid sequences of binding and non-binding VL domains demonstrated that amino acid residues crucial to the binding of protein L were distributed over a large area outside the hypervariable antigen-binding regions.
Akerström
B
B
Department of Medical and Physiological Chemistry, Lund University, Sweden.
Nilson
B H
BH
Hoogenboom
H R
HR
Björck
L
L
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002846:Chromatography, Affinity; D006801:Humans; D007128:Immunoglobulin Fragments / Q000378:metabolism*; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; C055047:Ig L-binding protein, Peptostreptococcus; C034284:IgG Fc-binding protein, Streptococcus; D007128:Immunoglobulin Fragments; D008565:Membrane Proteins; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; C055966:immunoglobulin Fv; C064006:protein H, Streptococcus