Document Detail


Rapid expression screening of eukaryotic membrane proteins in Pichia pastoris.
MedLine Citation:
PMID:  23339074     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Cory L Brooks; Melissa Morrison; M Joanne Lemieux
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2013-02-21
Journal Detail:
Title:  Protein science : a publication of the Protein Society     Volume:  22     ISSN:  1469-896X     ISO Abbreviation:  Protein Sci.     Publication Date:  2013 Apr 
Date Detail:
Created Date:  2013-03-20     Completed Date:  2013-08-26     Revised Date:  2014-04-01    
Medline Journal Info:
Nlm Unique ID:  9211750     Medline TA:  Protein Sci     Country:  United States    
Other Details:
Languages:  eng     Pagination:  425-33     Citation Subset:  IM    
Copyright Information:
Copyright © 2013 The Protein Society.
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MeSH Terms
Descriptor/Qualifier:
Animals
Aquaporin 4 / biosynthesis,  genetics
Biotechnology
Cloning, Molecular
Electrophoresis, Polyacrylamide Gel
Green Fluorescent Proteins / biosynthesis,  chemistry,  genetics,  metabolism
Humans
Membrane Proteins / biosynthesis*,  chemistry,  genetics,  metabolism
Mice
Phosphatidylethanolamine N-Methyltransferase / biosynthesis,  genetics
Pichia / genetics,  metabolism*
Protein Conformation
Recombinant Fusion Proteins / biosynthesis*,  chemistry,  genetics,  metabolism
Grant Support
ID/Acronym/Agency:
//Canadian Institutes of Health Research
Chemical
Reg. No./Substance:
0/AQP4 protein, human; 0/Aquaporin 4; 0/Membrane Proteins; 0/Recombinant Fusion Proteins; 147336-22-9/Green Fluorescent Proteins; EC 2.1.1.17/PEMT protein, mouse; EC 2.1.1.17/Phosphatidylethanolamine N-Methyltransferase
Comments/Corrections

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