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Publication Abstract from PubMed

The Nogo-66 receptor (NgR1) is part of a signaling complex that inhibits axon regeneration in the CNS. Truncated soluble versions of NgR1 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 leucine-rich repeat (LRR) regions in NgR1 are flanked by N- and C-terminal disulfide-containing "cap" domains (LRRNT and LRRCT, respectively). In this work we show that, although functionally active, the NgR1(310)-Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT, 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 NgR1 truncation site or the spacing between the NgR1 and Fc domains, or replacing cysteines within the NgR1 or IgG hinge regions. One variant, which incorporates subsititutions of Cys266 and Cys309 with alanines, completely eliminated disulfide scrambling, while maintaining functional in vitro and in vivo efficacy. This modified NgR1-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.

Resolution of disulfide heterogeneity in Nogo receptor 1 fusion proteins by molecular engineering.,Weinreb PH, Wen D, Qian F, Wildes CP, Garber EA, Walus L, Jung MY, Wang J, Relton JK, Amatucci J, Wang R, Porreca F, Silvian L, Meier W, Pepinsky RB, Lee DH Biotechnol Appl Biochem. 2010 Sep 3. PMID:20815818^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.