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Oxidative refolding of recombinant prochymosin
1Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
The Biochemical Journal
|May 7, 1999
Summary
Recombinant prochymosin refolding is more efficient with partially reduced forms, utilizing native structural elements. Disulphide bond formation and rearrangement are key steps in achieving the native conformation.
Area of Science:
- Biochemistry
- Protein Chemistry
- Molecular Biology
Background:
- Recombinant prochymosin is produced in Escherichia coli inclusion bodies.
- Solubilized prochymosin contains free thiol groups and disulphide bonds, existing in a partially reduced state.
Purpose of the Study:
- To investigate the disulphide-coupled refolding of recombinant prochymosin.
- To understand the role of native structural elements and disulphide bonds in renaturation.
- To propose a model for prochymosin refolding.
Main Methods:
- Solubilization of prochymosin from inclusion bodies.
- Renaturation experiments under different redox conditions (partially reduced vs. fully reduced).
- Addition of glutathione (GSH/GSSG) to facilitate disulphide rearrangement.
- Two-stage refolding process at different pH conditions (pH 11 and pH 8).
Main Results:
- Partially reduced prochymosin refolds more efficiently than fully reduced prochymosin.
- Native structural elements in inclusion bodies act as nuclei for correct refolding.
- Glutathione (GSH/GSSG) enhances renaturation, particularly for fully reduced prochymosin.
- A two-stage refolding model was proposed: pH 11 for disulphide bond formation/rearrangement and pH 8 for tertiary structure formation.
Conclusions:
- The refolding of recombinant prochymosin is a disulphide-coupled process.
- Optimized refolding conditions involve specific pH stages and redox environments.
- Disulphide rearrangement is a critical, rate-limiting step that can be facilitated by various agents.