Spectroscopic techniques for bioprocess optimization

Escherichia coli is often used as a host for recombinant protein production due 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 and novel promising solubilization methods have been developed that retain the existing native-like structure of the protein. In this context the key factors for increased yield of the bioactive product will be improved knowledge of the role of cultivation and expression conditions in IB formation and structural composition (quality) as well as down-stream functionalization related to the intrinsic properties of the protein. In terms of a holistic view of process control and application of novel methods for structural monitoring yield can be optimized. In the present project a set of spectroscopic techniques which provide complementary structural information and allow protein structure monitoring in the whole process will be applied: Fourier transform infrared microscopy (FTIR), (attenuated total reflectance) FTIR-, vibrational circular dichroism- (VCD), automated IR microplate reader, electronic circular dichroism-(ECD), fluorescence- and UV-Vis-spectroscopy. Novel devices that allow live monitoring of IB protein solubilization will be tested. Four structurally well characterized proteins (horseradish peroxidase C, interleukin-2, subtilisin and b-lactamase) known to be produced as IBs in E. coli were selected because their heterologous expression and bioanalysis is well established at the two Departments involved in this project. Since refolding strongly depends on the intrinsic properties of the target protein the un- and refolding pathway(s) of these four model proteins will be investigated by multimixing stopped-flow analyses (CD, UV-Vis, fluorescence) and time-resolved FTIR spectroscopy in order to elucidate productive refolding pathways that circumvent the collapse of secondary structures (aggregation). With this information the rational design of solubilization regimes that retain the secondary structure in IBs will be achieved.