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Expression of multisubunit proteins in Leishmania tarentolae
1Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
Methods in Molecular Biology (Clifton, N.J.)
|December 14, 2011
Summary
Leishmania tarentolae offers an efficient system for producing complex recombinant proteins, like human laminin-332, with proper folding and modifications. This protozoan system rivals mammalian cells for producing functional glycoproteins.
Area of Science:
- Biotechnology and Recombinant Protein Expression
- Cell Biology and Molecular Genetics
Background:
- Mammalian cell expression systems are preferred for recombinant proteins requiring post-translational modifications but suffer from low efficiency.
- Alternative systems like Escherichia coli and yeast offer higher efficiency but lack complex eukaryotic modifications.
- A novel expression system using Leishmania tarentolae combines easy handling with eukaryotic folding and mammalian-type post-translational modifications.
Purpose of the Study:
- To evaluate the Leishmania tarentolae expression system for producing recombinant human laminin (LM)-332, a large heterotrimeric glycoprotein.
- To demonstrate the system's capability for efficient multi-gene expression and secretion of complex proteins.
Main Methods:
- Development of a recombinant Leishmania tarentolae strain for expressing the three subunits of human LM-332.
- Cultivation of the recombinant strain to facilitate heterotrimer formation and secretion.
- Purification of the recombinant LM-332 (rLM-332) from the culture medium.
- Assessment of rLM-332's cell adhesion activity to confirm proper folding and assembly.
Main Results:
- The Leishmania tarentolae system successfully produced a recombinant strain that efficiently formed and secreted heterotrimeric LM-332.
- Purified rLM-332 exhibited cell adhesion activity comparable to that produced in mammalian cells.
- This indicates successful proper folding and assembly of the complex glycoprotein in the protozoan system.
Conclusions:
- The Leishmania tarentolae expression system is a viable and efficient platform for producing complex, post-translationally modified recombinant proteins like human LM-332.
- This system provides a valuable alternative to traditional mammalian cell expression, offering improved efficiency and ease of handling.
- The study provides a detailed protocol for multiple gene expression in L. tarentolae, facilitating its broader application.
