Endonuclease is a nuclease (nucleic acid degrading enzyme) which hydrolyzes the phosphodiester bond of a polynucleotide chain. Endonuclease recognizes and binds to a specific nucleotide sequence along DNA molecules, whereby molecules within the recognition sequence is cut. Endonuclease is a requisite enzyme for today's advanced gene engineering techniques for cloning and analyzing genes.
A site-specific endonuclease Endo.SceI (hereinafter, also referred to as "SceI") from an eucaryotic microorganism (e.g., yeast) is known to be a heterodimer having subunits of 75 kDa and 50 kDa. The subunits of SceI as well as genes encoding the subunits have been cloned, and the nucleotide sequences thereof have been determined (for 75 kDa subunit, see Morishima, N. et al., J. Biol. Chem. 265, 15189-15197 (1990) and for 50 kDa subunit, see JP-B-7-77556).
In order to widely utilize the above-described endonuclease for artificially modifying a biochemical agent, a gene or the like, the endonuclease needs to be mass-produced with a gene expression system. The endonuclease does not function unless it recognizes a specific nucleotide sequence, i.e., the endonuclease needs to be specific to the nucleotide sequence to be recognized.
The 50 kDa subunit of the above-described endonuclease SceI is encoded by mitochondrial genomes of yeast (Saccharomyces cerevisiae). A gene of a mitochondrial genome of yeast contains codons unique to mitochondria which are different from amino acid codons (universal codons) used in gene expression systems from organisms generally used for mass expression of protein (E.coli, baculovirus, yeast, etc.). If this gene of the mitochondrial genome is directly used, the protein expression system hardly produces a protein of an original amino acid sequence. For example, while TGA is a stop codon as a universal codon, it is a different codon coding for other amino acid (Trp) in mitochondria. A gene may be normally expressed in mitochondria but expression of the same gene may not result in a complete protein in a general expression system such as E.coli due to incomplete translation caused by the stop codon.
USPTO Backgrounds
USPTO Backgrounds