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Detection of Protein Ubiquitination
Published on: August 19, 2009
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Intein-ubiquitin chimeric domain for coordinated protein coexpression
Bei Zhang1, Zhenlin Han1, Sandeep Kumar2
1Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI, USA.
Journal of Biotechnology
|August 16, 2019
Summary
This study introduces a novel intein-ubiquitin system for efficiently coexpressing multiple proteins from a single gene in eukaryotic cells. This method ensures accurate protein release and sequence integrity, advancing biotechnology applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- Efficient coproduction of multiple proteins is crucial for life sciences and biotechnology.
- Current methods for coexpressing multiple proteins can be complex and may lead to altered protein function.
Purpose of the Study:
- To develop a novel system for coordinated coexpression of multiple proteins in eukaryotic hosts.
- To leverage the synergy between an engineered mini-intein and a ubiquitin variant for protein release.
Main Methods:
- A single open reading frame encoding a polyprotein precursor with proteins of interest (POIs) linked by an intein-ubiquitin fusion domain was engineered.
- Intein-mediated autocatalytic cleavage and deubiquitinase-mediated cleavage were utilized for POI release.
- Fluorescent reporter proteins were used for proof-of-concept validation in yeast and plants.
Main Results:
- The intein-ubiquitin system demonstrated effective coexpression and release of multiple proteins from a single precursor.
- Essentially complete release of POIs was confirmed via western blot analysis.
- Proteins expressed using this system maintained their intended sequences, preserving function.
Conclusions:
- The engineered intein-ubiquitin system provides an efficient and precise method for coproducing multiple proteins in eukaryotic systems.
- This approach offers a valuable tool for various applications in biotechnology and synthetic biology.
- The system ensures protein sequence integrity, critical for maintaining biological function.
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