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Many mammalian receptor domains, among them a large number of potential therapeutic target proteins, are highly aggregation-prone upon heterologous expression in bacteria. This severely limits functional studies of such receptor domains and also their engineering towards improved properties. One of these proteins is the Nogoreceptor, which plays a central role in mediating the inhibition of axon growth and functional recovery after injury of the adult mammalian central nervous system. We show here that the ligand binding domain of the Nogoreceptor folds to an active conformation in ternary ribosomal complexes, as formed in ribosome display. In these complexes the receptor is still connected, via a C-terminal tether, to the peptidyl tRNA in the ribosome and the mRNA also stays connected. The ribosome prevents aggregation of the protein, which aggregates as soon as the release from the ribosome is triggered. In contrast, no active receptor was observed in phage display, where aggregation appears to prevent incorporation of the protein into the phage coat. This strategy sets the stage for rapidly studying defined mutations of such aggregation-prone receptors in vitro and to improve their properties by in vitro evolution using the ribosome display technology.

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Originally published in: Protein Folding Handbook. Part II. Edited by Johannes Buchner and Thomas Kiefhaber. Copyright © 2005 Wiley‐VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3‐527‐30784‐2 The sections in this article are Introduction Kinetic and Thermodynamic Aspects of Natural Proteins The Stability of Natural Proteins Different Kinds of “Stability” Thermodynamic Stability Kinetic Stability Folding Efficiency The Engineering Approach Consensus Strategies Principles Examples Structure‐based Engineering Entropic Stabilization Hydrophobic Core Packing Charge Interactions Hydrogen Bonding Disallowed Phi‐Psi Angles Local Secondary Structure Propensities Exposed Hydrophobic Side Chains Inter‐domain Interactions Case Study: Combining Consensus Design and Rational Engineering to Yield Antibodies with Favorable Biophysical Properties The Selection and Evolution Approach Principles Screening and Selection Technologies Available for Improving Biophysical Properties In Vitro Display Technologies Partial in Vitro Display Technologies In Vivo Selection Technologies Selection for Enhanced Biophysical Properties Selection for Solubility Selection for Protein Display Rates Selection on the Basis of Cellular Quality Control Selection for Increased Stability General Strategies Protein Destabilization Selections Based on Elevated Temperature Selections Based on Destabilizing Agents Selection for Proteolytic Stability Conclusions Acknowledgements

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