Authors: Daniel Hopkins Sujatha Gomathinayagam Heather Lynaugh Terrance A Stadheim Stephen R Hamilton
Publish Date: 2014/02/14
Volume: 98, Issue: 6, Pages: 2573-2583
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 humanlike N and Olinked 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 twoamino acid peptide from the Nterminus 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 Nterminal 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
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