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Peptide mapping of subtilisins as a practical tool for locating protein sequence errors during extensive protein
1COGNIS, Incorporated, Santa Rosa, California 95407.
Analytical Biochemistry
|November 15, 1994
Summary
This study presents a rapid method for protein sequence verification after mutagenesis. The technique uses acid treatment, urea denaturation, and HPLC peptide mapping to detect even minor protein alterations, aiding protein engineering.
Area of Science:
- Biochemistry
- Protein Engineering
- Analytical Chemistry
Background:
- Rational protein design involves iterative amino acid substitutions.
- Accurate and rapid verification of protein sequences post-mutagenesis is crucial.
Purpose of the Study:
- To develop a sensitive and fast method for checking protein sequences after mutagenesis.
- To apply this method for quality control in protein engineering.
Main Methods:
- Subtilisin BL was subjected to acid treatment and urea denaturation.
- Enzyme degradation by trypsin.
- Peptide separation and mapping using reversed-phase High-Performance Liquid Chromatography (HPLC).
Main Results:
- The method rapidly generated peptide maps differentiating conservative amino acid alterations.
- Identified erroneous mutations in engineered proteins.
- Quantified active protease concentration against a standard.
- Measured methionine oxidation rates, specifically at methionine-216.
Conclusions:
- This HPLC-based peptide mapping is a robust technique for verifying protein sequences in protein engineering.
- The method enables accurate assessment of protein integrity and specific modifications.