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Method for the production of polypeptides
| Details |
Inventors: Egel-Mitani, Michi; Brandt, Jakob; Vad, Knud;
Assignee: Novo Nordisk A/S (Bagsv.ae butted.rd, DK)
Primary Examiner: Schwartzman; Robert A.
Assistant Examiner: Sandals; William
Attorney, Agent or Firm: Zelson, Esq.; Steve T., Green, Esq.; Reza
The present invention relates to a novel method for the production of short chain polypeptides, including polypeptides having up to 3 disulfide bonds and/or structures rich in basic amino acid residues, and open structured short chain polypeptides, e.g. glucagon, glucagon like peptides and their functional analogues, in genetically modified yeast cells, said genetically modified yeast cells, and a method for the preparation of said yeast cells. |
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DETAILED DESCRIPTION The above identified purpose is achieved with the method according to the present invention which comprises culturing a yeast which has reduced activity of Yap3 protease (Yap3p) or a homologue thereof and has been transformed with a hybrid vector comprising a yeast promoter operably linked to a DNA sequence coding for a polypeptide having up to 55 amino acids, preferably from 10-50 amino acids, preferably from 15-40, or from 25-35 amino acids in the polypeptide chain, and having from 0 to 3 disulphide bonds, preferably no more than one disulphide bond in the structure, such as glucagon or glucagon like peptides, and isolating said polypeptides.
Preferably, the yeast cells lack Yap3p activity through disruption of the YAP3 gene.
Using a YAP3 disrupted yeast strain for the production of polypeptides having from 1-70 amino acids, preferably from 1-40, and more preferably from 10-30 amino acids in the polypeptide chain, and having no more than one disulphide bonds in the structure such as polypeptides encoded by the glucagon precursor gene including glucagon, GRPP, GLP-1, GLP-2, and their functional analogues thereof result in a remarkably improved yield of up to about 2-fold and even 10-fold compared to the yield from the corresponding YAP3 wild-type yeast strain.
It has been found that using a YAP3 disrupted yeast strain for the production of heterologous polypeptides having up to 55 amino acids, preferably from 10-50 amino acids, preferably from 15-40 or from 25-35 amino acids, in the polypeptide chain, and having from 0 to 3 disulphide bonds, preferably no more than one disulphide bond in the structure, such as polypeptides encoded by the glucagon precursor gene including glucagon, GRPP, GLP-1, GLP-2, and their functional analogues thereof or CRF, e.
g.
as shown in SEQ ID NO:3 herein, or truncated and/or N-terminally extended forms of CART, preferably EEID-CART.
sub.
55-102 (SEQ ID NO:2), result in a remarkably improved yield of the heterologous polypeptide of up to about 2-fold and even 10-fold compared to the yield obtained from the corresponding YAP3 wild-type yeast strain
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Drugs 
