University of Natural Resources and Life Sciences, Vienna (BOKU) - Research portal
Gewählte Doctoral Thesis:
Gerd Margreiter
(2008):
The impact of cultivation conditions on bacterial inclusion bodies.
Doctoral Thesis,
BOKU-Universität für Bodenkultur,
pp 126.
UB BOKU
obvsg
- Abstract:
- The efficiency of the renaturation process is a key issue in recombinant protein production. Structure and size of inclusion bodies (IBs) are important parameters in this process and are therefore the main objective of this thesis. The impact of cultivation conditions on the structure of IBs was investigated by means of FT-IR spectroscopy and assessed by refolding experiments. However, almost no variation of the secondary structure of the model proteins Npro-GFP and TEM1-beta-lactamase associated with changes of cultivation conditions could be detected in IBs. Moreover, aggregates formed during refolding of IBs previously dissolved in urea showed very similar FT-IR spectra suggesting (i) the existence of only one critical folding step from which either aggregation (IB formation) or native folding branches off, and (ii) underlining the important role of the amino acid sequence in aggregation. For extraction and solubilization of IBs their size and density plays a key role for process optimization. In order to characterize the inclusion body attributes sedimentation field-flow fractionation was applied. For this purpose beta-lactamase IBs from various fed-batch cultivations were investigated. It could be demonstrated that induction strategies have a strong impact on size distribution. The full-induction process delivered Gaussian size distribution of IBs whereby part-induction processes resulted in a broad size distribution at the end of the cultivation. By observation of IB size distributions in the course of the production phase deeper insight into IB formation kinetics could be gained. Sedimentation field-flow fractionation thereby proved to be a powerful tool for IB size determination. Further the aptitude of FT-IR spectroscopy and chemometrics for fast IB quantification was investigated. It could be shown that the applicability of this technique depends on the structure of the IBs.