Protein Engineering, Vol. 15, No. 11, 931-941,
November 2002
© 2002 Oxford University Press
An engineered IN-1 Fab fragment with improved affinity for the Nogo-A axonal growth inhibitor permits immunochemical detection and shows enhanced neutralizing activity
1 Lehrstuhl für Biologische Chemie, Technische Universität München, Freising-Weihenstephan, Germany, 3 Institut für medizinische Chemie und Biochemie, Universität Innsbruck, Austria and 4 Institut für Hirnforschung, Universität und Dept. Biologie, ETH, Zürich, Switzerland
The myelin axonal growth inhibitor NI-220/250 (Nogo-A) has attracted considerable attention in elucidating the mechanisms that account for the lack of plasticity in the adult central nervous system. The cognate monoclonal antibody IN-1, which was obtained prior to the molecular characterization of its Nogo-A antigen, has played a crucial role in this respect. However, this murine IgM/
antibody does not only provide an inappropriate format for in vivo studies, its low antigen affinity has also hampered the thorough structure–function analysis of its neutralizing effect toward the Nogo-A inhibitor on a molecular basis. We describe here the affinity maturation of a bacterially produced functional IN-1 Fab fragment via protein engineering. A soluble fragment of Nogo-A derived from the central exon 3 of its gene, which was prepared by secretion into the periplasm of Escherichia coli, served as a target in these experiments. After repeated cycles of site-directed random mutagenesis and screening, the mutant II.1.8 of the IN-1 Fab fragment was obtained, carrying five side chain substitutions within CDR-L3. Its dissociation constant for the complex with the recombinant Nogo-A fragment was determined in surface plasmon resonance measurements as approximately 1 µM. The affinity of the unmutated IN-1 Fab fragment was 8-fold lower. The engineered Fab fragment appeared to be well suited for the specific detection of Nogo-A in immunochemical assays and for the histochemical staining of myelin-rich tissue sections. Most importantly, its concentration-dependent neutralizing effect on the Nogo-A inhibitory activity was significantly enhanced in cell culture. This study confirms Nogo-A to be the antigen of the IN-1 antibody and it demonstrates increased potential of the engineered Fab fragment as a reagent for promoting axonal regeneration in vivo.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Schlapschy, I. Theobald, H. Mack, M. Schottelius, H.-J. Wester, and A. Skerra Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life Protein Eng. Des. Sel., June 26, 2007; (2007) gzm020v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Nasreen, M. Vogt, H. J. Kim, A. Eichinger, and A. Skerra Solubility engineering and crystallization of human apolipoprotein D Protein Sci., January 1, 2006; 15(1): 190 - 199. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Schlapschy, H. Gruber, O. Gresch, C. Schafer, C. Renner, M. Pfreundschuh, and A. Skerra Functional humanization of an anti-CD30 Fab fragment for the immunotherapy of Hodgkin's lymphoma using an in vitro evolution approach Protein Eng. Des. Sel., December 1, 2004; 17(12): 847 - 860. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Breithaupt, A. Schubart, H. Zander, A. Skerra, R. Huber, C. Linington, and U. Jacob Structural insights into the antigenicity of myelin oligodendrocyte glycoprotein PNAS, August 5, 2003; 100(16): 9446 - 9451. [Abstract] [Full Text] [PDF]
|
|