Purification of antibodies using protein L-binding framework structures in the light chain variable domain.
Protein L from the bacterial species Peptostreptococcus magnus binds specifically to the variable domain of Ig light chains, without interfering with the antigen-binding site. In this work a genetically engineered fragment of protein L, including four of the repeated Ig-binding repeat units, was employed for the purification of Ig from various sources. Thus, IgG, IgM, and IgA were purified from human and mouse serum in a single step using protein L-Sepharose affinity chromatography. Moreover, human and mouse monoclonal IgG, IgM, and IgA, and human IgG Fab fragments, as well as a mouse/human chimeric recombinant antibody, could be purified from cultures of hybridoma cells or antibody-producing bacterial cells, with protein L-Sepharose. This was also the case with a humanized mouse antibody, in which mouse hypervariable antigen-binding regions had been introduced into a protein L-binding kappa subtype III human IgG. These experiments demonstrate that it is possible to engineer antibodies and antibody fragments (Fab, Fv) with protein L-binding framework regions, which can then be utilized in a protein L-based purification protocol.
Nilson
B H
BH
Department of Medical and Physiological Chemistry, University of Lund, Sweden.
Lögdberg
L
L
Kastern
W
W
Björck
L
L
Akerström
B
B
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D006801:Humans; D007147:Immunoglobulin Light Chains / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D010215:Papio; D010462:Peptostreptococcus / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism
D000906:Antibodies; D001426:Bacterial Proteins; C055047:Ig L-binding protein, Peptostreptococcus; D007147:Immunoglobulin Light Chains; D011993:Recombinant Fusion Proteins