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Primary structure of Mucor miehei aspartyl protease: evidence for a zymogen intermediate
Abstract:
The gene encoding the aspartyl protease of the filamentous fungus Mucor miehei has been cloned in Escherichia coli and the DNA sequenced. The deduced primary translation product contains an N-terminal region of 69 amino acid (aa) residues not present in the mature protein. By analogy to the evolutionarily related mammalian gastric aspartyl proteases it is inferred that the primary secreted product is a zymogen containing a 47-aa propeptide. This propeptide is presumably removed in the later steps of the secretion process or upon secretion into the medium. To study the effects of modifications of the protease structure on its maturation by enzyme-engineering methods, an efficient expression system was sought. In E. coli, transcription of the preproenzyme coding sequence from a bacterial promoter results primarily in the accumulation of unsecreted, enzymatically inactive polypeptides, immunologically related to the authentic protease. In Aspergillus nidulans expression of the cloned gene, probably from its own promoter, results in the secretion into the culture medium of polypeptides which, compared to the authentic protease, are similar in specific activity, but differ in the character of their asparagine-linked oligosaccharides.
Insights
The gene for Mucor miehei aspartyl protease was cloned and sequenced. Expression in Aspergillus nidulans secreted active protease, unlike E. coli which produced inactive forms, aiding enzyme engineering studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Mycology
Background:
- The aspartyl protease from Mucor miehei is a significant enzyme with potential industrial applications.
- Understanding its gene structure and expression is crucial for protein engineering and production.
Purpose of the Study:
- To clone and sequence the gene encoding the Mucor miehei aspartyl protease.
- To investigate heterologous expression systems for protease maturation and enzyme engineering.
Main Methods:
- Gene cloning and DNA sequencing of the Mucor miehei aspartyl protease.
- Heterologous expression in Escherichia coli and Aspergillus nidulans.
- Analysis of secreted and unsecreted polypeptide products and their enzymatic activity.
Main Results:
- The gene was successfully cloned and sequenced, revealing a preproenzyme with a signal peptide and a propeptide.
- Expression in E. coli resulted in inactive, unsecreted polypeptides.
- Expression in A. nidulans led to secretion of active protease with altered oligosaccharide modifications.
Conclusions:
- A. nidulans provides a suitable system for secreting active Mucor miehei aspartyl protease, facilitating enzyme engineering.
- Differences in expression between E. coli and A. nidulans highlight the importance of host systems for protein processing.