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Two-chain bacteriorhodopsin synthesized by Schizosaccharomyces pombe
O K Hansen1, M Pompejus, H J Fritz
1Institut für Molekulare Genetik, Georg-August-Universität Göttingen, Germany.
Biological Chemistry Hoppe-Seyler
|October 1, 1994
Summary
Researchers engineered functional bacteriorhodopsin (BR), a light-driven proton pump, in yeast. This was achieved by expressing two separate gene fragments that spontaneously assembled into the complete, functional protein.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- Bacteriorhodopsin (BR) is a light-driven proton pump found in Halobacterium salinarium.
- Functional BR production is crucial for various biotechnological applications.
Purpose of the Study:
- To develop a method for producing functional bacteriorhodopsin in a eukaryotic system.
- To investigate the in vivo assembly of a two-chain bacteriorhodopsin protein.
Main Methods:
- Simultaneous expression of two separate structural genes in the fission yeast Schizosaccharomyces pombe.
- One gene encoded an aminoterminal BR fragment (first two transmembrane helices).
- The other gene encoded the remaining part of the protein.
Main Results:
- Functional, two-chain bacteriorhodopsin was successfully produced in Schizosaccharomyces pombe.
- The expressed fragments spontaneously assembled in vivo to form functional BR.
- Purification of the functional BR was achieved using immobilized metal ion affinity chromatography.
Conclusions:
- Simultaneous expression of split genes in yeast is a viable strategy for producing functional bacteriorhodopsin.
- This method facilitates the production and purification of a complex membrane protein.