ArticleMicrobial cell factories2016
Liquid PTVA: a faster and cheaper alternative for generating multi-copy clones in Pichia pastoris.
Article in Microbial cell factories, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 19 papers.
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Who cites it
19 citing papers in PubMed.
- Enhanced secretion of an Agrocybe aegerita peroxygenase variant in K. phaffii using the native signal peptide.AMB Express · 2026Article
- Recent advances in microbial fermentation for the production of leghemoglobin.World journal of microbiology & biotechnology · 2025Review
- Advancing recombinant protein expression in Komagataella phaffii: opportunities and challenges.FEMS yeast research · 2025Review
- Customizable and stable multilocus chromosomal integration: a novel glucose-dependent selection system in Aureobasidium spp.Biotechnology for biofuels and bioproducts · 2024Article
- Enhancing the expression of the unspecific peroxygenase in Komagataella phaffii through a combination strategy.Applied microbiology and biotechnology · 2024Article
- Harnessing alkaline-pH regulatable promoters for efficient methanol-free expression of enzymes of industrial interest in Komagataella Phaffii.Microbial cell factories · 2024Article
- Review
- Alternative PCR-Based Approaches for Generation ofMicroorganisms · 2023Article
- Full-length versus truncated α-factor secretory signal sequences for expression of recombinant human insulin precursor in yeast Pichia pastoris: a comparison.Journal, genetic engineering & biotechnology · 2023Article
- The fluorescent protein iLOV as a reporter for screening of high-yield production of antimicrobial peptides in Pichia pastoris.Microbial biotechnology · 2022Article
- Development of synthetic biology tools to engineerSynthetic and systems biotechnology · 2021Review
- A New Cold-Active Glucose Oxidase From Penicillium: High-Level Expression and Application in Fish Preservation.Frontiers in microbiology · 2020Article
- CRISPR-Cas9-mediated genomic multiloci integration in Pichia pastoris.Microbial cell factories · 2019Article
- Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris.Applied and environmental microbiology · 2018Article
- Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris.AMB Express · 2017Article
- Molecular strategies to increase the levels of heterologous transcripts in Komagataella phaffii for protein production.Bioengineered · 2017Review
- Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker.Microbial cell factories · 2017Article
- A Mitochondrial Autonomously Replicating Sequence fromFrontiers in microbiology · 2017Article
- Production of Chimeric Acidic α-Amylase by the RecombinantFrontiers in microbiology · 2017Article
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2 authors.
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Abstract
backgroundMultiple cognate gene copy clones have often been used in order to increase the yield of recombinant protein expression in the yeast Pichia pastoris. The method of posttransformational vector amplification (PTVA) has allowed for the efficient generation of multi-copy clones in P. pastoris. However, despite its relative ease and success, this process can be expensive and time consuming.
resultsWe have developed a modified version of PTVA, called Liquid PTVA, which allows for faster and cheaper selection of multi-copy clones. Cultures are grown in liquid medium with only a final selection carried out on agar plates, reducing overall antibiotic usage and increasing the speed of clone amplification. In addition, it was established that starting PTVA with a single copy clone resulted in higher copy number strains for both traditional plate PTVA and liquid PTVA. Furthermore, using the Zeocin selection marker in liquid PTVA results in strains with higher growth rates, which could be beneficial for recombinant protein production processes.
conclusionsWe present a methodology for creating multi-copy clones that can be achieved over 12 days instead of the traditional 45 and at approximately half the cost.
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