Resolution of disulfide heterogeneity in Nogo receptor I fusion proteins by molecular engineering.

Paul H. Weinreb, Dingyi Wen, Fang Qian, Craig P. Wildes, Ellen A. Garber, Lee Walus, Mi young Jung, Joy Wang, Jane K. Relton, Joseph Amatucci, Ruizhong Wang, Frank Porreca, Laura Silvian, Werner Meier, R. Blake Pepinsky, Daniel H.S. Lee

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

NgRI (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgRI have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgRI are flanked by N- and C-terminal disulfide-containing 'cap' domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgRI(310)-Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgRI truncation site or the spacing between the NgRI and Fc domains, or replacing cysteines within the NgRI or IgG hinge regions. One variant, which incorporates replacements of Cys2 and Cys3 with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgRI-Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins.

Original languageEnglish (US)
Pages (from-to)31-45
Number of pages15
JournalBiotechnology and applied biochemistry
Volume57
Issue number1
DOIs
StatePublished - Sep 2010
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Molecular Medicine
  • Biomedical Engineering
  • Applied Microbiology and Biotechnology
  • Drug Discovery
  • Process Chemistry and Technology

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  • Cite this

    Weinreb, P. H., Wen, D., Qian, F., Wildes, C. P., Garber, E. A., Walus, L., Jung, M. Y., Wang, J., Relton, J. K., Amatucci, J., Wang, R., Porreca, F., Silvian, L., Meier, W., Pepinsky, R. B., & Lee, D. H. S. (2010). Resolution of disulfide heterogeneity in Nogo receptor I fusion proteins by molecular engineering. Biotechnology and applied biochemistry, 57(1), 31-45. https://doi.org/10.1042/BA20100061