Grafting of protein L-binding activity onto recombinant antibody fragments.
Recombinant antibody fragments consisting of variable domains can be easily produced in various host cells, but there is no universal system that can be used to purify and detect them in the free form or complexed with their antigen. Protein L (PpL) is a cell wall protein isolated from Peptostreptococcus magnus, which has been reported to interact with the V-KAPPA chain of some, but not all, antibodies. Here we grafted the V-KAPPA framework region 1 (FR1) sequence of a high-affinity PpL-binding antibody onto single-chain antibody fragments (scFvs), which have no reactivity with PpL. This substitution made it possible to purify and detect scFvs using PpL conjugates. It did not hinder scFv folding and expression in recombinant bacteria, and it did not interfere with their antigen-binding function. We also identified residue 12 as being potentially able to alter PpL binding. This study, therefore, suggests a way of engineering a PpL-binding site on any scFv without interfering with its function. This could provide a universally applicable method both for the rapid purification of functional recombinant antibody fragments and for their detection even when complexed with their antigen without requiring fusion to an epitope Flag.
Muzard
Julien
J
Muséum national d'Histoire naturelle, CNRS FRE 3206-CP39, Paris cedex 05, France.
Adi-Bessalem
Sonia
S
Juste
Matthieu
M
Laraba-Djebari
Fatima
F
Aubrey
Nicolas
N
Billiald
Philippe
P
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D019295:Computational Biology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D007128:Immunoglobulin Fragments; C116593:L-protein, Peptococcus magnus; D011994:Recombinant Proteins