Method for making acylated polypeptides
AssigneeNovo Nordisk A/S
InventorsIb Jonassen, Michi Egel-Mitani, Per Balschmidt, Jan Markussen, Ivan Diers, Thomas Borglum Kjeldsen
Life of this patent
Estimating the term…
Abstract
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 ε-amino groups.
Claims44
A method for making an acylated polypeptide, wherein said polypeptide comprises at least one lysine residue that is acylated on its ε-amino group, said method comprising:
- (i) expressing in a suitable host cell a precursor of said polypeptide, wherein said precursor comprises said polypeptide and an N-terminal extension, said N-terminal extension being cleavable from the polypeptide at a lysine cleavage site;
- (ii) preferentially acylating the ε-amino group of said at least one lysine residue in the polypeptide without acylating the ε-amino group of the lysine cleavage site, to produce an acylated precursor;
- and
- (ii) removing the N-terminal extension from the acylated precursor by enzymatic cleavage to produce said acylated polypeptide.
A precursor for a polypeptide, wherein said precursor has the formula
- N-terminal-extension-Lys-Z3-Z4-*polypeptide*,
- wherein Lys is a cleavage site, the N-terminal extension has 2-14 amino acid residues and is capable of preventing or minimizing acylation of the Lys cleavage site and protecting the polypeptide precursor against proteolytic degradation during production of the precursor, Z3 is His or Tyr and is the N-terminal amino acid residue in the desired polypeptide, Z4 is the next amino acid residue from the N-terminal end in the desired polypeptide and is Ala, Ser or Gly, and *polypeptide* is the remaining sequence of the desired polypeptide.
Dependencies are inferred from the claim text.