Online monitoring of protein refolding in inclusion body processing using intrinsic fluorescence.

Inclusion bodies (IBs) are protein aggregates formed as a result of overexpression of recombinant protein in E. coli. The formation of IBs is a valuable strategy of recombinant protein production despite the need for additional processing steps, i.e., isolation, solubilization and refolding. Industrial process development of protein refolding is a labor-intensive task based largely on empirical approaches rather than knowledge-driven strategies. A prerequisite for knowledge-driven process development is a reliable monitoring strategy. This work explores the potential of intrinsic tryptophan and tyrosine fluorescence for real-time and in situ monitoring of protein refolding. In contrast to commonly established process analytical technology (PAT), this technique showed high sensitivity with reproducible measurements for protein concentrations down to 0.01 g L - 1 . The change of protein conformation during refolding is reflected as a shift in the position of the maxima of the tryptophan and tyrosine fluorescence spectra as well as change in the signal intensity. The shift in the peak position, expressed as average emission wavelength of a spectrum, was correlated to the amount of folding intermediates whereas the intensity integral correlates to the extent of aggregation. These correlations were implemented as an observation function into a mechanistic model. The versatility and transferability of the technique were demonstrated on the refolding of three different proteins with varying structural complexity. The technique was also successfully applied to detect the effect of additives and process mode on the refolding process efficiency. Thus, the methodology presented poses a generic and reliable PAT tool enabling real-time process monitoring of protein refolding.

Corrigendum: State-of-the-art and novel approaches to mild solubilization of inclusion bodies.

[This corrects the article DOI: 10.3389/fbioe.2023.1249196.].

State-of-the-art and novel approaches to mild solubilization of inclusion bodies.

Throughout the twenty-first century, the view on inclusion bodies (IBs) has shifted from undesired by-products towards a targeted production strategy for recombinant proteins. Inclusion bodies can easily be separated from the crude extract after cell lysis and contain the product in high purity. However, additional solubilization and refolding steps are required in the processing of IBs to recover the native protein. These unit operations remain a highly empirical field of research in which processes are developed on a case-by-case basis using elaborate screening strategies. It has been shown that a reduction in denaturant concentration during protein solubilization can increase the subsequent refolding yield due to the preservation of correctly folded protein structures. Therefore, many novel solubilization techniques have been developed in the pursuit of mild solubilization conditions that avoid total protein denaturation. In this respect, ionic liquids have been investigated as promising agents, being able to solubilize amyloid-like aggregates and stabilize correctly folded protein structures at the same time. This review briefly summarizes the state-of-the-art of mild solubilization of IBs and highlights some challenges that prevent these novel techniques from being yet adopted in industry. We suggest mechanistic models based on the thermodynamics of protein unfolding with the aid of molecular dynamics simulations as a possible approach to solve these challenges in the future.