| US 7,572,884 B2 | ||
| Method for making acylated polypeptides | ||
| Thomas Hoeg-Jensen, Klampenborg (Denmark); Michi Egel-Mitani, Vedbaek (Denmark); Per Balschmidt, Espergaerde (Denmark); Jan Markussen, Herlev (Denmark); and Ivan Diers, Vaerlose (Denmark) | ||
| Assigned to Novo Nordisk A/S, Bagsvaerd (Denmark) | ||
| Filed on Jul. 28, 2005, as Appl. No. 11/191,574. | ||
| Application 11/191574 is a continuation of application No. 10/205270, filed on Jul. 24, 2002, abandoned. | ||
| Claims priority of provisional application 60/310952, filed on Aug. 08, 2001. | ||
| Claims priority of application No. 2001 01140 (DK), filed on Jul. 24, 2001. | ||
| Prior Publication US 2005/0272125 A1, Dec. 08, 2005 | ||
| Int. Cl. A61K 38/00 (2006.01); A61K 38/04 (2006.01) | ||
| U.S. Cl. 530—345 [530/330; 530/329; 530/324; 514/2; 514/12] | 6 Claims |
| 1. A method for making GLP-1 or a GLP-1 analogue comprising at least one lysine residue being acylated in its ε-amino group,
said method comprising the following steps:
(i) culturing a host cell comprising a polynucleotide sequence encoding a precursor molecule of the GLP-1 or GLP-1 analogue
under suitable conditions for expression of the precursor molecule said precursor molecule comprising an N-terminal extension
capable of protecting the GLP-1 or GLP-1 analogue against proteolytic degradation, said N-terminal extension comprising: i)
a sequence of Glu-Glu-Ala-Glu(SEQ ID NO:27), and ii) a cleavage site positioned at its C-terminal end for cleavage from the
GLP-1 or GLP-1 analogue, wherein the cleavage site is not a Lys;
(ii) separating the expressed precursor from the culture broth;
(iii) acylating the ε-amino group of at least one lysine residue in the desired polypeptide;
(iv) removing the N-terminal extension by chemical and/or enzymatic cleavage and isolating the acylated polypeptide by suitable
means,
wherein said GLP-1 or GLP-1 analogue is a peptide of GLP-1(7-36) or GLP-1(7-37).
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