Method for making maturated insulin polypeptides in a fungal cell

The invention is related to a method for making mature human insulin or an analogue thereof by culturing a fungi cell comprising a DNA sequence encoding a precursor for human insulin or an analogue of human insulin which precursor comprises the B-chain of human insulin or an analogue thereof, the A-chain of human insulin or an analogue thereof and a C-peptide linking the B-chain and the A-chain together thereof, wherein the C-peptide comprises at least one Kex2 cleavage site and an amino acid sequence attached at one end to the C-terminal amino acid residue in the B-chain and at the other end to the Kex2 site which amino acid sequence will facilitate a more efficient Kex2 cleavage within the fungi cell. The C-terminal extension of the B-chain may furthermore be capable of subsequently being cleaved off from the C-terminal amino acid residue in the B-chain by means of a carboxypeptidase activity either within the fungi cell or subsequently in the culture medium.