The formation of inclusion bodies (IBs) is the major bottleneck for both research and industrial application.High-level expression of recombinant proteins in bacterial host systems significantly reduces the recovery of functional proteins. To improve the quality and yield of functional protein, a better understanding of unfolding and refolding of peptibody is required. This investigation is focused on understanding the unfolding and refolding of the multi-disulfide-containing peptide-Fc fusion protein, a peptibody expressed in E. coli. The mature peptibody is a homodimer. Each chain refolds to form two intra-chain disulfide linkages, and the two chains are connected in the neck region of the Fc portion by two disulfide bridges. For quantification of refolded peptibody, we have utilized different orthogonal techniques like, RP-HPLC. The unfolding behavior of peptibody was analyzed by Nano-DSC, mass spectrometry, and fluorescence spectroscopy at different concentrations of solubilizing reagent at different time points and temperatures. The analysis of experimental denaturation curves helps us understand the thermodynamic parameters of peptibody unfolding such as ΔG, ΔTm, ΔH, ΔCp. All analysis of in-house peptibody is performed in comparison with the innovator molecule. The obtained data is essential for further structural, functional studies and possible therapeutic applications.

Understanding unfolding and refolding of peptibody

ACS Spring 2022

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