Spectroscopic techniques for bioprocess optimization

Escherichia coli is often used as a host for recombinant protein production due to its simple genetic manipulation, established strategies for recombinant protein expression, rapid growth and physiological knowledge. High level expression in E. coli often leads to an accumulation of the target protein in so-called inclusion bodies (IBs), which has been considered previously to be a problem, but is more and more acknowledged as a chance since it combines the advantages of a high accumulation of the target protein, efficient strategies for protein isolation and purification without the need of complicated coexpression systems. A major bottleneck of state of the art production regimes is the non-predictable and generally low yield of the renaturation step due to prominent unproductive side reactions leading to aggregation of the solubilized protein. Spectroscopic analysis clearly showed that IB proteins can exist at intermediate folding states with composition of secondary structure similar to the native forms. Recently, novel promising solubilization methods have been developed that retain the existing native-like structure of the protein and allow efficient non-denaturing solubilization with high yield of the bioactive product. However, cultivation and expression conditions influence the structural features of the target protein in the IBs. Thus, novel methods that allow structural monitoring of target proteins in the whole bioprocess are demanded. To cope with these complex problems a set of spectroscopic techniques (Fourier transform infrared (FTIR) microscopy, (attenuated total reflectance) FTIR-, vibrational circular dichroism- (VCD), automated IR microplate reader, electronic circular dichroism-(ECD), fluorescence- and UV-Vis-spectroscopy), which allow rapid elucidation of secondary and tertiary structural elements of proteins, will provide efficient and expedient tools. They will be used for the first time for monitoring the whole bioprocess including IB formation, solubilization and refolding. Three structurally well characterized proteins (horseradish peroxidase C, interleukin-2, and ß-lactamase), known to be produced as IBs in E. coli, were selected because their heterologous expression and bioanalysis is well established at the involved Departments. Since refolding strongly depends on the intrinsic properties of the target protein, the un- and refolding pathway(s) of these three model proteins will be investigated by multimixing stopped-flow analysis (CD, UV-Vis, fluorescence) and time-resolved FTIR spectroscopy in order to elucidate productive refolding pathways that circumvent the collapse of secondary structures (aggregation) by conventional methods. By combining these data with those obtained from bioprocess monitoring the rational design of solubilization regimes that retain the secondary structure in IBs and provide optimal yield of the bioactive product will be achieved.