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Published online before print May 28, 2004, 10.1110/ps.03540504
Protein Science (2004), 13:1882-1891. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Affinity transfer by CDR grafting on a nonimmunoglobulin scaffold

Magali Nicaise1, Marielle Valerio-Lepiniec1, Philippe Minard and Michel Desmadril

Laboratoire de Modélisation et d’Ingénierie des Protéines, UMR8619, Université de Paris-Sud, Bât 430, F-91405 Orsay Cedex, France

(RECEIVED December 2, 2003; FINAL REVISION April 8, 2004; ACCEPTED April 8, 2004)



Abstract

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.

Keywords: neocarzinostatin; CDR3 loop; camel antilysozyme; drug targeting

Abbreviations: CD, circular dichroism • CDR, complementarity determining region • ELISA, enzyme-linked immunosorbent assay • EtBr, ethidium bromide • Gdm-Cl, guanidinium chloride • HEPES, N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid • HEWL, hen egg white lysozyme • Ig, immunoglobulin • NCS, neocarzinostatin • NCS-CDR3, engineered neocarzinostatin containing the CDR3 loop of the VHH camel antilysozyme • SPR, surface plasmon resonance • TBS, Tris-buffered saline • Tris, Tris(hydroxymethyl) aminomethane • WT-NCS, wild-type neocarzinostatin • {Delta}Hcal, calorimetric enthalpy

Reprint requests to: Michel Desmadril, Laboratoire de Modélisation et d’Ingénierie des Protéines, UMR8619, Université de Paris-Sud, Bât 430, F-91405 Orsay Cedex, France; e-mail: michel.desmadril{at}mip.u-psud.fr; fax: 33-1-69-85-37-15.

1 These authors contributed equally to this work.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03540504.


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