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Targeted Deletion of Los1 Homologue Affects the Production of a Recombinant Model Protein in Pichia pastoris.
- Source :
-
Iranian biomedical journal [Iran Biomed J] 2021 Jul 01; Vol. 25 (4), pp. 255-64. Date of Electronic Publication: 2021 Jul 01. - Publication Year :
- 2021
-
Abstract
- Background: The methylotrophic yeast Pichia pastoris is an appealing production host for a variety of recombinant proteins, including biologics. In this sense, various genetic- and non-genetic-based techniques have been implemented to improve the production efficiency of this expression platform. Loss of supression (Los1) encodes a non-essential nuclear tRNA exporter in Saccharomyces cerevisiae, which its deletion extends replicative lifespan. Herein, a los1-deficient strain of P. pastoris was generated and characterized.<br />Methods: A gene disruption cassette was prepared and transformed into an anti-CD22-expressing strain of P. pastoris. A δ los1 mutant was isolated and confirmed. The drug sensitivity of the mutant was also assessed. The growth pattern and the level of anti-CD22 single-chain variable fragment (scFv) expression were compared between the parent and mutant strains.<br />Resuults: The los1 homologue was found to be a non-essential gene in P. pastoris. Furthermore, the susceptibility of los1 deletion strain to protein synthesis inhibitors was altered. This strain showed an approximately 1.85-fold increase in the extracellular level of anti-CD22 scFv (p < 0.05). The maximum concentrations of total proteins secreted by δ los1 and parent strains were 125 mg/L and 68 mg/L, respectively.<br />Conclusion: The presented data suggest that the targeted disruption of los1 homologue in P. pastoris can result in a higher expression level of our target protein. Findings of this study may improve the current strategies used in optimizing the productivity of recombinant P. pastoris strains.
- Subjects :
- Cell Survival physiology
Nuclear Pore Complex Proteins antagonists & inhibitors
Saccharomyces cerevisiae Proteins antagonists & inhibitors
Gene Deletion
Gene Targeting methods
Nuclear Pore Complex Proteins genetics
Recombinant Proteins genetics
Saccharomyces cerevisiae Proteins genetics
Saccharomycetales genetics
Subjects
Details
- Language :
- English
- ISSN :
- 2008-823X
- Volume :
- 25
- Issue :
- 4
- Database :
- MEDLINE
- Journal :
- Iranian biomedical journal
- Publication Type :
- Academic Journal
- Accession number :
- 33992037
- Full Text :
- https://doi.org/10.52547/ibj.25.4.255