Artificial linear episome-based protein expression system for protozoon Leishmania tarentolae

Area of Science:

• Molecular Biology
• Biotechnology
• Parasitology

Background:

• Leishmania tarentolae is a key model organism for tropical diseases and recombinant protein production.
• Existing expression systems require genomic integration, limiting flexibility.
• A need exists for alternative, stable, and efficient expression platforms.

Purpose of the Study:

• To develop a new expression architecture for Leishmania tarentolae based on linear episomal elements.
• To assess the stability, expression levels, and utility of this novel system for recombinant protein production.
• To demonstrate the system's applicability in a parallelized format for diverse protein targets.

Main Methods:

• Construction and propagation of linear expression vectors in E. coli.
• Conversion of circular plasmids to linear episomes with telomere-like structures.
• Transfection of Leishmania tarentolae and assessment of recombinant protein expression.
• Parallel expression and purification of multiple target proteins fused to EGFP.

Main Results:

• Achieved high-level overexpression of recombinant proteins (>10% of total cellular protein).
• Demonstrated stable propagation of linear elements in L. tarentolae for over 90 generations.
• Successfully expressed 30 out of 37 tested proteins using the linear vector system.
• Enabled parallelized protein production without the need for clonal selection.

Conclusions:

• The novel linear element-based expression system offers a stable and efficient platform for recombinant protein production in Leishmania tarentolae.
• This system overcomes limitations of genomic integration and facilitates high-yield, parallelized bioproduction.
• The findings significantly advance the utility of L. tarentolae as a host for biotechnological applications.