ArticleProtein science : a publication of the Protein Society2013
Rapid expression screening of eukaryotic membrane proteins in Pichia pastoris.
Article in Protein science : a publication of the Protein Society, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed, 28 citations in OpenAlex.
- Evaluation of heterologous expression in Pichia pastoris of Pine Weevil TRPA1 by GFP and flow cytometry.Microbial cell factories · 2024Article
- Heterologous (Over) Expression of Human SoLute Carrier (SLC) in Yeast: A Well-Recognized Tool for Human Transporter Function/Structure Studies.Life (Basel, Switzerland) · 2022Review
- Expression and Purification of Human Mitochondrial Intramembrane Protease PARL.Methods in molecular biology (Clifton, N.J.) · 2021Article
- Changes in Membrane Protein Structural Biology.Biology · 2020Review
- The topology of the ER-resident phospholipid methyltransferase Opi3 ofThe Journal of biological chemistry · 2020Article
- Smoothing membrane protein structure determination by initial upstream stage improvements.Applied microbiology and biotechnology · 2019Review
- Structural biology workflow for the expression and characterization of functional human sodium glucose transporter type 1 in Pichia pastoris.Scientific reports · 2019Article
- Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris.Applied and environmental microbiology · 2018Article
- Improved microscale cultivation ofFungal biology and biotechnology · 2018Article
- Article
- Restriction site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris.Microbial cell factories · 2015Article
- Method to increase the yield of eukaryotic membrane protein expression in Saccharomyces cerevisiae for structural and functional studies.Protein science : a publication of the Protein Society · 2014Article
- Comprehensive clone screening and evaluation of fed-batch strategies in a microbioreactor and lab scale stirred tank bioreactor system: application on Pichia pastoris producing Rhizopus oryzae lipase.Microbial cell factories · 2014Article
- Insights into the catalytic properties of the mitochondrial rhomboid protease PARL.The Journal of biological chemistryArticle
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
The overexpression of milligram quantities of protein remains a key bottleneck in membrane protein structural biology. A challenge of particular difficulty has been the overproduction of eukaryotic membrane proteins. In order to cope with the frequently poor expression levels associated with these challenging proteins, it is often necessary to screen a large number of homologues to find a well expressing clone. To facilitate this process using the heterologous, eukaryotic expression host Pichia pastoris, we have developed a simple fluorescent induction plate-screening assay that allows for the rapid detection of well expressing clones of eukaryotic membrane proteins that have been fused to GFP. Using a eukaryotic membrane protein known to express well in P. pastoris (human aquaporin 4) and homologues of the ER associated membrane protein phosphatidylethanolamine N-methyltransferase (PEMT), we demonstrate that when a large number of clones are screened, a small number of highly expressing "jackpot" clones can be isolated. A jackpot PEMT clone resulted in 5 mg/L yield after purification. The method allows for the facile simultaneous screening of hundreds of clones providing an alternate to in-culture screening and will greatly accelerate the search for overexpressing eukaryotic membrane proteins.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.