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Elimination of diaminopeptidase activity in Pichia pastoris for therapeutic protein production
- Source :
- Applied microbiology and biotechnology. 98(6)
- Publication Year :
- 2013
-
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.
- Subjects :
- Proteases
Glycosylation
Proteolysis
medicine.medical_treatment
Biology
Applied Microbiology and Biotechnology
Pichia
Pichia pastoris
chemistry.chemical_compound
Gene Knockout Techniques
medicine
Dipeptidyl-Peptidases and Tripeptidyl-Peptidases
chemistry.chemical_classification
Biological Products
Protease
medicine.diagnostic_test
Proteolytic enzymes
General Medicine
biology.organism_classification
Yeast
Recombinant Proteins
chemistry
Biochemistry
Glycoprotein
Peptides
Biotechnology
Subjects
Details
- ISSN :
- 14320614
- Volume :
- 98
- Issue :
- 6
- Database :
- OpenAIRE
- Journal :
- Applied microbiology and biotechnology
- Accession number :
- edsair.doi.dedup.....f5febb8ed766375150cad477c48d8ad3