Tuesday, November 6, 2007 - 2:08 PM
209e

Protein Refolding By Means Of Gradient Chromatography

Esteban Jaramillo Freydell1, Ottens Marcel2, Wielen Luuk2, and Michel Eppink3. (1) Department of Biotechnology, Delft University of Technology, Julianalaan 67, Delft, 2628 BC, Netherlands, (2) Biotechnology, Delft University of Technology, Julianalaan 67, Delft, 2628 BC, Netherlands, (3) DSP, Diosynth Biotechnology Europe, Oss, Netherlands

PROTEIN REFOLDING BY MEANS OF GRADIENT CHROMATOGRAPHY

The over expression of recombinant proteins in Escherichia coli leads in most cases to their accumulation in the form of insoluble aggregates referred to as inclusion bodies. The inclusion bodies are composed of partially folded intermediates, which in most cases have a native-like secondary structure. In order to release the product from them, the inclusion bodies must be solubilised in a buffer composition that most of the times include aggregation suppressors and redox agents. To obtain the native and bioactive product the concentration of the chemicals used for the solubilization have to be changed preferably in a controllable manner, minimizing the product lost through the competitive reactions, namely misfolding and aggregation. The industrial process generally used is dilution, which is an economical process but limited by the formation of native product and the equipment size required to cope with the product demand. Recent developments have indicated that application of chromatography for refolding may be successful. In this work we will experimentally and theoretically show column refolding in batch as well in continuous mode, making use of an SMB system, including urea and redox gradients in order to promote refolding of a fusion protein trapped in inclusion bodies.



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