For selective purification of proteins, the only separation technique used broadly in industry today is chromatography, which is characterized by low capacity and poor scaleability. It must also be preceded by unit operations like centrifugation, filtration and precipitation, leading to overall low process yields.
There is thus a strong need for alternative methods which are predictable, easily scaleable, and also selective, to produce pure proteins. Liquid-liquid extraction in aqueous two-phase systems presents these possibilities and has already been proven useful for some industrial separations. However, there is a need for further development of this technology for purification of recombinant proteins: selectivity in protein extraction needs to be increased, phase recycling needs to be improved for reduction of costs.
The proposed research concerns the development of generic technology for integrated bioprocesses for protein production and isolation. This will be done by considering the downstream processing of the target protein already at the genetic level. Genetic engineering will be used for fusion of peptide or protein tags to the target protein, taking care to retain the protein biological activity. The modifications will be exploited for designing selective extraction systems for the target proteins. Two industrially important target proteins, a cellulase (endoglucanase) and a lipase (cutinase) will be studied.
The project combines efforts of leading European research groups within the different areas described below, and involves the active participation of two biotechnological industries. The work is planned for multidisciplinary interaction, from genetic engineering and protein properties to phase partitioning and biochemical engineering, in order to design truly integrated and predictable bioprocesses:
- Genetic engineering. This will involve expression of target proteins with different modifications introduced by fusion of peptide or protein tags to the N- or C-terminus of the protein, with the aim to achieve enhanced partitioning and selectivity in aqueous two-phase extractions. The tags will differ in hydrophobicity and charge, and will include peptide tails, folded protein domains and a small protein.
- Protein partitioning in aqueous two-phase systems. Effect of tag fusion on protein partitioning will be studied in a variety of phase systems, including conventional polyethylene glycol (PEG)/salt, and recently developed novel polymer and detergent systems, where phase separation can be induced by temperature or pH change and phase components can be effectively recycled.
- Properties of modified proteins. The conformation of peptide tags and interactions of peptide tag with target protein will be studied by high field NMR. This will allow determination of the exposure of the tag to the phase forming polymers, and will help in the design of effective tags. Functional properties related to the activity and stability of the modified proteins will be studied.
- Biochemical engineering of aqueous two-phase extractions. Engineering and scale-up studies will be done on phase systems found optimal from the above studies, with special emphasis on partitioning and removal of DNA, recycling of phase forming chemicals under laboratory and pilot scale conditions. A final stage in the project will be large scale tests of recombinant protein purification in SOm3 scale in the production plant of one of the industrial partners.
Funding SchemeCSC - Cost-sharing contracts
100 44 Stockholm
3133 AT Vlaardingen