TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector.

Area of Science:

• Molecular Biology
• Synthetic Biology
• Biotechnology

Background:

• Efficient delivery and expression of multiple genes are crucial for synthetic biology applications like pathway engineering and stem cell induction.
• Conventional methods often suffer from inefficiency, nonstoichiometric expression, and limitations on the number of simultaneously expressed genes.

Purpose of the Study:

• To develop a versatile and efficient approach for simultaneous delivery and expression of multiple genes using a single expression vector.
• To overcome the limitations of conventional multi-gene expression systems.

Main Methods:

• Mimicking RNA virus protein expression strategies by creating a single fusion protein including genes of interest and TEV protease.
• Utilizing TEV protease's self-cleavage activity to release individual functional protein components from the fusion polyprotein.
• Demonstrating the method in E. coli using SDS-PAGE for analysis and in mammalian cells using differential localization and live-cell imaging.

Main Results:

• Efficient cleavage of the fusion protein into functional components was confirmed in E. coli.
• Consistent stoichiometry and distinctive subcellular targeting of processed products were observed in mammalian cells.
• Successful expression and separation of up to three genes were achieved in both E. coli and mammalian systems using the single TEV protease self-processing vector.

Conclusions:

• The developed TEV protease self-processing vector system offers an efficient and stoichiometric method for multi-gene delivery and expression.
• This versatile approach is applicable in both prokaryotic (E. coli) and eukaryotic (mammalian) cells.
• The system provides a valuable tool for advancing synthetic biology and other applications requiring precise control over multiple gene expression.