[0001] The invention relates to a polynucleotide encoding an oxygen-sensitive protein and a mitochondrial targeting peptide which can be expressed in a eukaryotic cell under aerobic conditions. The invention also refers to a method for expressing said oxygen-sensitive protein in a eukaryotic cell as well as to a method forin vitroreconstitution of active nitrogenase protein complex.
1. A polynucleotide encoding a fusion protein which comprises an oxygen-sensitive protein and a mitochondrial targeting peptide. 2. The polynucleotide according to claim 1, wherein said prokaryotic organism isAzotobacter vinelandii. 3. The polynucleotide according to claims 1 or 2, wherein said oxygen-sensitive protein is NifH. 4. The polynucleotide according to claims 1 to 3 wherein said mitochondrial targeting peptide is N-terminal to the oxygen-sensitive protein. 5. The polynucleotide according to any of claims 1 to 4, wherein said mitochondrial targeting peptide comprises the mitochondrial targeting peptide of superoxide dismutase. 6. The polynucleotide according to any of claims 1 to 5 wherein the sequences encoding the fusion protein have been codon optimized for expression in a eukaryotic cell. 7. The polynucleotide according to any of claims 1 to 6 further comprising transcriptional regulatory elements operatively linked to said polynucleotide or translational regulatory elements operatively linked to the fusion protein encoded by said polynucleotide. 8. The polynucleotide according to any of claims 1 to 7, wherein if the oxygen-sensitive protein is NifH, then the polynucleotide further comprises a nucleotide sequence which encodes a fusion protein comprising the NifM protein and a second mitochondrial targeting peptide. 9. The polynucleotide according to claim 8 wherein the sequences encoding the fusion protein comprising the NifM protein have been codon optimized for expression in a eukaryotic cell. 10. An expression vector comprising the polynucleotide according to any of claims 1 to 9. 11. A eukaryotic cell comprising the polynucleotide according to any of claims 1 to 16 or an expression vector according to claim 10. 12. The eukaryotic cell according to claim 11, wherein said eukaryotic cell is aSaccharomyces cerevisiaecell. 13. Method for expressing an oxygen-sensitive protein in a eukaryotic cell comprising the steps of: (i) introducing into said cell a polynucleotide according to any of claims 1 to 9 or a vector according to claim 10, (ii) growing said cell under conditions allowing the expression of said oxygen-sensitive protein and, if desired, (iii) purifying said oxygen-sensitive protein under anaerobic conditions. 14. Method forin vitroreconstitution of active nitrogenase protein complex comprising the steps of: i) introducing into said cell a polynucleotide according to any of claims 1 to 19 or a vector according to claim 10, wherein the oxygen-sensitive protein is NifH and wherein the cell further comprises a nucleotide sequence which encodes a fusion protein comprising the NifM protein and a mitochondrial targeting peptide wherein said second mitochondrial targeting peptide is N-terminal to NifM, ii) growing said cell under conditions allowing the expression of NifH and NifM proteins, iii) purifying NifH protein from said cells under anaerobic conditions and iv) contacting said purified NifH of step (iii) with the NifDK complex. 15. The method of claim 14, wherein the NifH/NifDK molar ratio in step iv) is of at least 10.