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Production of crystallizable human chymase from a Bacillus subtilis system
M E McGrath1, A E Osawa, M G Barnes
1Arris Pharmaceutical, South San Francisco, CA 94080, USA. mcgrath@arris.com
FEBS Letters
|August 25, 1997
Summary
Bacillus subtilis efficiently produces human mast cell chymase, a serine protease. This engineered bacterial system is valuable for expressing various proteins, with optimized crystal structures achieved for further study.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Bacillus subtilis is a well-established host for protein expression.
- Human mast cell chymase is a key serine protease involved in inflammatory responses.
- Efficient production and structural characterization of chymase are crucial for therapeutic development.
Purpose of the Study:
- To establish Bacillus subtilis as a viable expression system for human mast cell chymase.
- To optimize the purification and crystallization of recombinant chymase.
- To determine the crystal structures of PMSF-treated chymase.
Main Methods:
- Utilized a Bacillus subtilis strain deficient in extracellular proteases for recombinant protein production.
- Employed multiple chromatography steps for protein purification.
- Optimized crystallization conditions and collected diffraction data for two distinct crystal forms.
Main Results:
- Achieved production of human mast cell chymase at 0.3-0.5 mg/l.
- Successfully obtained two crystal forms of PMSF-treated chymase: C2 (diffracting to 2.1 A) and P212121 (diffracting to 1.9 A).
- Characterized the unit cell dimensions and molecular content of each crystal form.
Conclusions:
- The engineered Bacillus subtilis strain is a suitable system for producing human mast cell chymase.
- The determined crystal structures provide a foundation for understanding chymase function and developing inhibitors.
- This work facilitates further structural and functional studies of serine proteases.