What question did this study set out to answer?

Establish a reliable heterologous expression system for bovine pancreatic ribonuclease A to facilitate protein engineering and assess the catalytic role of the conserved Gln11 residue.

January 1, 1995Protein Engineering Design and Selection

Engineering ribonuclease A: production, purification and characterization of wild-type enzyme and mutants at Gln11

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Authors

SDStephen B. delCardayréUniversity of British Columbia MRMarc RibóVall d'Hebron Institut de Recerca EYErich M. YokelUniversity of Wisconsin–Madison

Discussion

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Implication

Experimental study demonstrates high-yield bacterial expression of ribonuclease A and reveals Gln11 prevents non-productive substrate binding, highlighting key mechanisms in enzyme efficiency.

Key Points

Establish a reliable heterologous expression system for bovine pancreatic ribonuclease A to facilitate protein engineering and assess the catalytic role of the conserved Gln11 residue.
Cloned bovine pancreatic RNase A cDNA into expression plasmids for heterologous production in Saccharomyces cerevisiae and Escherichia coli.
Purified secreted enzyme from yeast and refolded bacterial enzyme from insoluble inclusion bodies via oxidation of reduced and denatured protein.
Generated Gln11 mutants (Ala, Asn, His) in E. coli and analyzed binding and turnover kinetics with natural and synthetic substrates.
S. cerevisiae secreted an active, highly glycosylated form of RNase A at a yield of 1 mg/l of culture.
E. coli expression coupled with in vitro oxidative refolding produced active enzyme at a yield of 50 mg/l of culture.
Analysis of Gln11 substitutions demonstrated that the primary functional role of Gln11 is to prevent non-productive substrate binding.

Cite This Study

delCardayré et al. (1995) studied this question.

synapsesocial.com/papers/6a92c27329f7ca37161f77a7 https://doi.org/10.1093/protein/8.3.261

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January 1, 1995Protein Engineering Design and Selection

Engineering ribonuclease A: production, purification and characterization of wild-type enzyme and mutants at Gln11

View Full Paper

Ask AI

Bookmark

Authors

SDStephen B. delCardayréUniversity of British Columbia MRMarc RibóVall d'Hebron Institut de Recerca EYErich M. YokelUniversity of Wisconsin–Madison

Discussion

Member takes

Implication

Experimental study demonstrates high-yield bacterial expression of ribonuclease A and reveals Gln11 prevents non-productive substrate binding, highlighting key mechanisms in enzyme efficiency.

Key Points

Establish a reliable heterologous expression system for bovine pancreatic ribonuclease A to facilitate protein engineering and assess the catalytic role of the conserved Gln11 residue.
Cloned bovine pancreatic RNase A cDNA into expression plasmids for heterologous production in Saccharomyces cerevisiae and Escherichia coli.
Purified secreted enzyme from yeast and refolded bacterial enzyme from insoluble inclusion bodies via oxidation of reduced and denatured protein.
Generated Gln11 mutants (Ala, Asn, His) in E. coli and analyzed binding and turnover kinetics with natural and synthetic substrates.
S. cerevisiae secreted an active, highly glycosylated form of RNase A at a yield of 1 mg/l of culture.
E. coli expression coupled with in vitro oxidative refolding produced active enzyme at a yield of 50 mg/l of culture.
Analysis of Gln11 substitutions demonstrated that the primary functional role of Gln11 is to prevent non-productive substrate binding.

Cite This Study

delCardayré et al. (1995) studied this question.

synapsesocial.com/papers/6a92c27329f7ca37161f77a7 https://doi.org/10.1093/protein/8.3.261

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