Novo Nordisk A/s

Asser Sloth Andersen

Jesper F Lau

Peter Madsen

Svend Havelund

Ib Jonassen

Jan Markussen

John Halstrom

Patrick William Garibay

Thomas Kruse

Paw Bloch

Ivan Verner Diers

Claus Ulrich Jessen

Jens Christian Norrild

Caspar Christensen

Per Noergaard

Inga Sig Nielsen Nørby

Inger Lautrup-Larsen

Per Nørgaard

Lars Hojlund Christensen

John Halstro Slashedm

Anders Robert Sørensen

Jesper Færgeman Lau

John Broberg Halstrom

Per NØRGAARD

Inga Sig Nielsen NØRBY

Lars Højlund Christensen

A semi-recombinant method for the production of GLP-1 analogues and derivatives with non-proteogenic amino acids in the N-terminal part combining the use of recombinant expression techniques and chemical peptide synthesis.

Semi-recombinant preparation of GLP-1 analogues

The invention is related to a method for making human insulin analogs by culturing an fungi cell comprising a DNA vector encoding a precursor for human insulin analog, wherein the said precursor comprises a connecting peptide flanked with cleavage sites at both junctions with the A- and the B-chain of the insulin peptide, respectively said cleavage sites being cleaved within the fungi cell allowing the cell to secrete high amount of correctly processed, mature two chain human insulin analog to the culture media.

Method for making mature insulin polypeptides

The invention provides an improved method for producing polypeptides with a C-terminal glycine in a yeast transformant being characterized in having a non functional KEX1 gene. The method is in particular well suited to produce polypeptides with an aromatic amino acid residue attached to the C-terminal glycine. The yeast strain may have further non-functional protease genes selected from PEP4, YPS1, MKCI, YPS3, YPS5, YPS6, YPS7, PRB1, STE13 and KEX2.

Method for making polypeptides

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.

Method for making maturated insulin polypeptides in a fungal cell

The present invention relates to protracted human insulin derivatives in which the A21 and the B3 amino acid residues are, independently, any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys; Phemay be deleted; the B30 amino acid residue is (a) a non-codable, lipophilic amino acid having from 10 to 24 carbon atoms, in which case an acyl group of a carboxylic acid with up to 5 carbon atoms is bound to the &#x3b5;-amino group of Lys; or (b) the B30 amino acid residue is deleted or is any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys, in any of which cases the &#x3b5;-amino group of Lyshas a lipophilic substituent; and any Zncomplexes thereof with the proviso that when B30 is Thr or Ala and A21 and B3 are both Asn, and Pheis present, then the insulin derivative is always present as a Zncomplex.

Acylated insulin

A process for producing high amount of proteins or polypeptides in yeast is disclosed. The process makes use of the CIT1 yeast promoter or a functional part or variant thereof. Examples of polypeptides which are expressed in high yields are insulin or insulin analogues or GLP1.

Production of heterologous polypeptides in yeast

The present invention relates to protracted human insulin derivatives in which the A21 and the B3 amino acid residues are, independently, any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys; Phe.sup.B1 may be deleted; the B30 amino acid residue is (a) a non-codable, lipophilic amino acid having from 10 to 24 carbon atoms, in which case an acyl group of a carboxylic acid with up to 5 carbon atoms is bound to the .epsilon.-amino group of Lys.sup.B29 ; or (b) the B30 amino acid residue is deleted or is any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys, in any of which cases the .epsilon.-amino group of Lys.sup.B29 has a lipophilic substituent; and any Zn.sup.2+ complexes thereof with the proviso that when B30 is Thr or Ala and A21 and B3 are both Asn, and Phe.sup.B1 is present, then the insulin derivative is always present as a Zn.sup.2+ complex.

The present invention relates to human insulin derivatives having a protracted profile of action in which the A21 and B3 amino acid residues are, independently, any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys; Phe.sup.B1 may be deleted; the B30 amino acid residue is (a) a non-codable, lipophilic amino acid having from 10 to 24 carbon atoms in which case an acyl group of a carboxylic acid with up to 5 carbon atoms is bound to the E-amino group of Lys.sup.B29 ; (b) any amino acid residue which can be coded by the genetic code except Lys, Arg and Cys, in which case a lipophilic substituent is bound to the E-amino group of Lys.sup.B29 ; or (c) deleted, in which case a lipophilic substituent is bound to the E-amino group of LyS.sup.B29 ; and any Zn.sup.2+ complexes thereof; provided that when the B30 amino acid residue is Thr or Ala, the A21 and B3 amino acid residues are both Asn and Phe.sup.B1 is present, then the insulin derivative is a Zn.sup.2 + complex.

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Herlev, Denmark

Asser Sloth Andersen