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Generation of Highly Specific Proteolytic Biocatalysts by Screening Technologies
T V Bobik1, N N Kostin2, V D Knorre2
1Laboratory of Biocatalysis, M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Organic Biochemistry, Russian Academy of Sciences, Moscow, Russia. bobik_tanya@mail.ru.
Bulletin of Experimental Biology and Medicine
|July 14, 2018
Summary
We developed a novel yeast display system to screen proteolytic enzymes. This method uses a substrate on yeast cells; cleavage releases a biotin-binding center, enabling fluorescent detection and selection of target proteinases with high specificity.
Area of Science:
- Biotechnology
- Enzymology
- Molecular Biology
Background:
- Proteolytic enzymes are crucial in biological processes.
- Screening for enzymes with specific cleavage activity is challenging.
- Existing methods lack efficiency and specificity.
Purpose of the Study:
- To develop a yeast display system for efficient screening of proteolytic enzymes.
- To enable selection of proteinases with predefined substrate specificities.
- To demonstrate the system's effectiveness using human enterokinase light chain.
Main Methods:
- Yeast display of substrate protein with a cleavage sequence.
- Cleavage releases a biotin-binding center.
- Fluorescent detection and cytofluorometry-based cell sorting for enzyme selection.
Main Results:
- The system effectively screens proteolytic enzyme libraries.
- High specificity selection of enzymes based on cleavage sequence was achieved.
- Human enterokinase light chain was successfully used as a model proteinase.
Conclusions:
- The proposed yeast display system is a powerful tool for discovering and engineering proteolytic enzymes.
- This method offers high throughput and specificity for enzyme screening.
- The system has broad applicability in enzyme discovery and protein engineering.
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