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Bacterial expression and refolding of human trypsinogen
Hubertus Hohenblum1, Karola Vorauer-Uhl, Hermann Katinger
1Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
Journal of Biotechnology
|April 6, 2004
Summary
Researchers achieved high-level expression of human trypsinogen 1 in E. coli using a T7 expression system. This method enabled efficient refolding and activation, proving useful for mammalian cell detachment in culture.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- Recombinant protein expression in E. coli often results in insoluble aggregates.
- Mammalian trypsinogens are challenging to express in active forms.
Purpose of the Study:
- To achieve high-level expression of human trypsinogen 1 in E. coli.
- To develop an efficient refolding and activation protocol for recombinant human trypsinogen 1.
- To demonstrate the utility of the expressed trypsinogen for cell culture applications.
Main Methods:
- Utilized the T7 expression system in E. coli.
- Employed an N-terminal fusion strategy with T7 protein 10.
- Optimized a refolding procedure with continuous feed of denatured protein.
- Achieved autocatalytic activation to generate active trypsin.
Main Results:
- Obtained high-level expression of human trypsinogen 1 (200 mg/g bacterial dry mass) via N-terminal fusion.
- Developed an optimized refolding protocol maintaining a 20-35% yield of active protein.
- Demonstrated successful conversion to active trypsin and its efficacy in detaching mammalian cells.
Conclusions:
- N-terminal fusion with T7 protein 10 enables high-level expression of human trypsinogen 1 in E. coli.
- An optimized refolding and activation strategy yields active trypsin suitable for biotechnological applications.
- Recombinant human trypsinogen 1 is a viable tool for mammalian cell detachment in culture.