Patent ID: 8691530

Claim:
A process for obtaining aspart insulin comprising the steps of: i) transforming a Pichia pastoris yeast strain with at least two expression vectors containing different selectable markers, each vector comprising a DNA construct encoding an aspart insulin precursor of formula I, wherein said formula I is Asp B28 , B(1-30)-X1-Y-X2-A(1-21) comprising the sequence identified as SEQ ID NO.6, wherein B(1-30) represents the complete human insulin B chain, in which the amino acid proline B28 was replaced by an aspart acid, X1 is Arg, Y is Trp, X2 is Arg and A(1-21) represents the human insulin A chain; ii) selecting from the strains transformed in step i), those MUT S recombinant strains containing more than 5 copies of the DNA construct integrated into the yeast genome; iii) selecting from the recombinant strains from step ii) above, the strain having the highest number of copies and having the highest expression level of the aspart insulin precursor iv) fermenting said recombinant strain; v) separating the aspart insulin precursor from the culture medium by chromatography, vi) enzymatically digesting said precursor under conditions suitable for converting at least 70% of aspart insulin precursor into mature aspart insulin; and vii) purifying the aspart insulin obtained from step (vi) by conventional chromatography.