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Recombinant human renin produced in different expression systems: biochemical properties and 3D structure
S Mathews1, H Döbeli, M Pruschy
1Pharma Division, Preclinical Research, F. Hoffmann-La Roche, Ltd., Basel, Switzerland.
Protein Expression and Purification
|February 1, 1996
Summary
This study shows that producing human prorenin, not mature renin, yields higher enzyme amounts in cell cultures. The 3D structures of both renin forms were identical, revealing key insights into enzyme activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Human renin is a key enzyme in the renin-angiotensin system, regulating blood pressure.
- Understanding renin's structure and production is crucial for developing effective therapeutics.
Purpose of the Study:
- To compare the expression levels of human mature renin versus human prorenin in different cell systems.
- To determine the 3D structure of expressed human renin and its relationship to prorenin.
- To investigate the structural impact of different expression strategies and glycosylation on renin.
Main Methods:
- Gene constructs for mature renin and preprorenin were used for expression in Sf9 and CHO cells.
- Enzyme purification of both mature renin and proteolytically derived active renin.
- Cocrystallization with the renin inhibitor Ro 42-5892.
- 3D structure determination using X-ray crystallography.
Main Results:
- Prorenin expression resulted in significantly higher enzyme yields compared to mature renin expression in both cell systems.
- The 3D structures of directly expressed mature renin and proteolytically derived active renin were determined.
- Despite variations in glycosylation and N-termini, the solved structures demonstrated identity.
Conclusions:
- Expressing human renin as preprorenin is a more efficient strategy for high-level enzyme production.
- The structural identity of different renin forms provides a basis for understanding enzyme function and inhibitor design.