Affinity transfer by CDR grafting on a nonimmunoglobulin scaffold.
Neocarzinostatin (NCS) is a small "all beta" protein displaying the same overall fold as immunoglobulins. This protein possesses a well-defined hydrophobic core and two loops structurally equivalent to the CDR1 and CDR3 of immunoglobulins. NCS is the most studied member of the enediynechromoprotein family, and is clinically used as an antitumoral agent. NCS has promise as a drug delivery vehicle if new binding specificities could be conferred on its protein scaffold. Previous studies have shown that the binding specificity of the crevasse can be extended to compounds completely unrelated to the natural enediyne chromophore family. We show here that it is possible to introduce new interaction capacities to obtain a protein useful for drug targeting by modifying the immunoglobulin CDR-like loops. We transferred the CDR3 of the VHH chain of camel antilysozyme immunoglobulin to the equivalent site in the corresponding loop of neocarzinostatin. We then evaluated the stability of the resulting structure and its affinity for lysozyme. The engineered NCS-CDR3 presents a structure similar to that of the wild-type NCS, and is stable and efficiently produced. ELISA, ITC, and SPR measurements demonstrated that the new NCS-CDR3 specifically bound lysozyme.
Nicaise
Magali
M
Laboratoire de Modélisation et d'Ingénierie des Protéines, UMR8619, Université de Paris-Sud, Bât 430, F-91405 Orsay Cedex, France.
Valerio-Lepiniec
Marielle
M
Minard
Philippe
P
Desmadril
Michel
M
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002162:Camelus; D022801:Complementarity Determining Regions / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016503:Drug Delivery Systems; D009113:Muramidase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics / Q000276:immunology; D009353:Zinostatin / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D022801:Complementarity Determining Regions; D011993:Recombinant Fusion Proteins; D009353:Zinostatin; D009113:Muramidase