Construction of an expression system for large oligomeric proteins

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We have constructed a protein expression system by using Dictyostelium discoideum, eukaryotic organisms living as single cells. For expressing oligomeric proteins in Dictyostelium cells, it is necessary to use multiple selection markers since one marker is required for expressing a single gene. We have used the blasticidin S deaminase for the new selection marker in Dictyostelium cells. The actin 15 promoter and the actin 6 terminator were fused to the Bsr gene in frame to drive its expression. Since the neomycin-resistance gene has been already in use, we can now use two selection markers to express at least two genes at the same time. We used this new expression system to express one myosin heavy chain and two light chains at the same time in Dictyostelium cells. In this way, we are now under way to express large amount of the head domain of Dictyostelium myosin for crystallization.