title: Elimination of diaminopeptidase activity in Pichia pastoris for therapeutic protein production.
abstract: Yeast are important production platforms for the generation of recombinant proteins. Nonetheless, their use has been restricted in the production of therapeutic proteins due to differences in their glycosylation profile with that of higher eukaryotes. The yeast strain Pichia pastoris is an industrially important organism. Recent advances in the glycoengineering of this strain offer the potential to produce therapeutic glycoproteins with sialylated human-like N- and O-linked glycans. However, like higher eukaryotes, yeast also express numerous proteases, many of which are either localized to the secretory pathway or pass through it en route to their final destination. As a consequence, nondesirable proteolysis of some recombinant proteins may occur, with the specific cleavage being dependent on the class of protease involved. Dipeptidyl aminopeptidases (DPP) are a class of proteolytic enzymes which remove a two-amino acid peptide from the N-terminus of a protein. In P. pastoris, two such enzymes have been identified, Ste13p and Dap2p. In the current report, we demonstrate that while the knockout of STE13 alone may protect certain proteins from N-terminal clipping, other proteins may require the double knockout of both STE13 and DAP2. As such, this understanding of DPP activity enhances the utility of the P. pastoris expression system, thus facilitating the production of recombinant therapeutic proteins with their intact native sequences.
authors: GlycoFi, Inc. (a wholly owned subsidiary of Merck & Co., Inc.), Biologics Discovery, Merck Research Laboratories, 16 Cavendish Court, Lebanon, NH, 03766, USA.
authors: Daniel
authors: D
authors: Hopkins
authors: Sujatha
authors: S
authors: Gomathinayagam
authors: Heather
authors: H
authors: Lynaugh
authors: Terrance A
authors: TA
authors: Stadheim
authors: Stephen R
authors: SR
authors: Hamilton
mesh_terms: D001688:Biological Products / Q000378:metabolism*; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000235:genetics* / Q000378:metabolism; D055786:Gene Knockout Techniques; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001688:Biological Products; D010455:Peptides; D011994:Recombinant Proteins; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases