| Autor: |
Nora Tir, Lina Heistinger, Clemens Grünwald-Gruber, Leo A. Jakob, Stephan Dickgiesser, Nicolas Rasche, Diethard Mattanovich |
| Jazyk: |
angličtina |
| Rok vydání: |
2022 |
| Předmět: |
|
| Zdroj: |
Microbial Cell Factories, Vol 21, Iss 1, Pp 1-16 (2022) |
| Druh dokumentu: |
article |
| ISSN: |
1475-2859 |
| DOI: |
10.1186/s12934-022-01882-6 |
| Popis: |
Abstract Background Expansion of the genetic code is a frequently employed approach for the modification of recombinant protein properties. It involves reassignment of a codon to another, e.g., unnatural, amino acid and requires the action of a pair of orthogonal tRNA and aminoacyl tRNA synthetase modified to recognize only the desired amino acid. This approach was applied for the production of trastuzumab IgG carrying p-azido-l-phenylalanine (pAzF) in the industrial yeast Pichia pastoris. Combining the knowledge of protein folding and secretion with bioreactor cultivations, the aim of the work was to make the production of monoclonal antibodies with an expanded genetic code cost-effective on a laboratory scale. Results Co-translational transport of proteins into the endoplasmic reticulum through secretion signal prepeptide change and overexpression of lumenal chaperones Kar2p and Lhs1p improved the production of trastuzumab IgG and its Fab fragment with incorporated pAzF. In the case of Fab, a knockout of vacuolar targeting for protein degradation further increased protein yield. Fed-batch bioreactor cultivations of engineered P. pastoris strains increased IgG and IgGpAzF productivity by around 50- and 20-fold compared to screenings, yielding up to 238 mg L−1 and 15 mg L−1 of fully assembled tetrameric protein, respectively. Successful site-specific incorporation of pAzF was confirmed by mass spectrometry. Conclusions Pichia pastoris was successfully employed for cost-effective laboratory-scale production of a monoclonal antibody with an unnatural amino acid. Applying the results of this work in glycoengineered strains, and taking further steps in process development opens great possibilities for utilizing P. pastoris in the development of antibodies for subsequent conjugations with, e.g., bioactive payloads. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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