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Critical analysis of lysozyme refolding kinetics
A Mark Buswell1, Anton P J Middelberg
1Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, UK.
Biotechnology Progress
|June 8, 2002
Summary
A new pathway for lysozyme refolding and aggregation was discovered, showing native protein can incorporate into aggregates. This finding challenges existing models, impacting industrial protein refolding reactor design.
Area of Science:
- Biochemistry
- Chemical Engineering
- Protein Science
Background:
- Existing kinetic models for lysozyme refolding often overestimate yield at high concentrations.
- These models are limited for industrial reactor design due to inaccuracies at relevant concentrations.
Purpose of the Study:
- To investigate the limitations of current lysozyme refolding kinetic models.
- To experimentally determine if refolded native lysozyme can be incorporated into aggregates during the refolding process.
Main Methods:
- Utilized a competing first- and third-order reaction scheme for kinetic analysis.
- Incorporated fluorescein isothiocyanate (FITC)-labeled native lysozyme into the refolding buffer.
- Analyzed aggregates for fluorescence to confirm incorporation of labeled protein.
Main Results:
- Fluorescence was detected in collected aggregates, confirming the incorporation of labeled native lysozyme.
- The experimental results indicate a pathway for native protein to join aggregates during refolding.
- The existing kinetic model was found to be not globally applicable.
Conclusions:
- The study experimentally validates the existence of a native protein to aggregate pathway during lysozyme refolding.
- Existing kinetic models that neglect this pathway can lead to suboptimal reactor design.
- Critical assessment of refolding kinetic models is essential before implementation in reactor design.