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Tobacco protein separation by aqueous two-phase extraction.
Deepa Balasubramaniam1, Carol Wilkinson, Kevin Van Cott
1Department of Biological Systems Engineering, 210 Seitz Hall, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.
Journal of Chromatography. A
|March 19, 2003
Summary
Aqueous two-phase extraction (ATPE) effectively purifies recombinant proteins from tobacco biomass. This protein-friendly method offers a scalable solution for downstream processing challenges, achieving high yields and purity.
Area of Science:
- Biotechnology
- Protein Purification
- Plant Molecular Farming
Background:
- Tobacco is a valuable host for producing high-value recombinant proteins.
- Large biomass quantities pose downstream processing challenges.
- Aqueous two-phase extraction (ATPE) is a scalable, protein-friendly purification technique.
Purpose of the Study:
- To evaluate ATPE for recombinant protein purification from tobacco.
- To optimize ATPE conditions for lysozyme purification from tobacco extract.
- To determine the partition behavior of tobacco proteins and model lysozyme.
Main Methods:
- ATPE using poly(ethylene glycol) (PEG)-salt systems.
- Fractional factorial designs to study PEG molecular mass, PEG concentration, salt concentration, and pH.
- Response surface methodology for optimizing selectivity and yield.
- Lysozyme as a model recombinant protein.
Main Results:
- PEG-sodium sulfate system showed high suitability for lysozyme purification.
- Optimized conditions achieved high selectivity of lysozyme over native tobacco proteins.
- Predicted lysozyme yield of 87%, purification factor of 4, and concentration factor of 14.
Conclusions:
- ATPE is a suitable method for initial recovery and partial purification of recombinant proteins from transgenic tobacco.
- The study demonstrates the efficacy of ATPE in overcoming biomass processing challenges.
- Optimized ATPE parameters enhance purification efficiency and yield.