Novo Nordisk A/s

Thomas Borglum Kjeldsen

Svend Ludvigsen

Per Balschmidt

Ib Jonassen

Jan Markussen

Niels Christian Kaarsholm

Michi Egel-Mitani

Ivan Verner Diers

Helle Fabricius Woldike

Knud Vad

Annette Frost Pettersson

The present invention related to a method of producing polypeptides in transformed host cells by expressing a precursor molecule of the desired polypeptide which are to be acylated in a subsequent in vitro step. The invention is also related to DNA-sequences, vectors and transformed host cells for use in the claimed method. Further, the present invention is related to certain precursors of the desired polypeptides and certain acylation methods. The invention provides a method for making polypeptides being preferentially acylated in certain lysine &#x3b5;-amino groups.

Method for making acylated polypeptides

Novel insulin precursors and insulin precursor analogs comprising a connecting peptide (mini C-peptide) of preferably up to 15 amino acid residues and comprising at least one Gly are provided. The precursors can be converted into human insulin or a human insulin analog. The precursors will typically have a distance between B27 (atom CG2) and A1 (atom CA) of less than 5 &#x212b;.

Method for making insulin precursors and insulin precursor analogues having improved fermentation yield in yeast

Novel insulin precursors comprising a connecting peptide (mini C-peptide) of preferably up to 6 amino acid residues and comprising at least one Pro are provided. The precursors can be converted into human insulin or desB30 human insulin.

Method for making human insulin precursors

Novel insulin precursors and insulin precursor analogs having a mini C-peptide comprising at least one aromatic amino acid residue have an increased folding stability. The novel insulin precursors and insulin precursor analogs can be expressed in yeast in high yields and are preferably not more 15 amino acid residues in length. Also provided are polynucleotide sequences encoding the claimed precursors or precursor analogs, and vectors and cell lines containing such polynucleotide sequences.

Method for making insulin precursors and insulin precursor analogs

Trypsin (trypsinogen) may be produced in a filamentous fungus by transforming a filamentous fungus with a vector comprising a DNA sequence encoding protrypsin or a derivative thereof N-terminally fused to a DNA sequence encoding a signal peptide, culturing the transformed filamentous fungus in a suitable culture medium to produce trypsinogen and recovering trypsinogen and/or trypsin from the medium.

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Virum, Denmark

Thomas Borglum Kjeldsen