title: Rapid expression screening of eukaryotic membrane proteins in Pichia pastoris.
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.
authors: Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors: Cory L
authors: CL
authors: Brooks
authors: Melissa
authors: M
authors: Morrison
authors: M
authors: M
authors: Joanne Lemieux
mesh_terms: D000818:Animals; D051401:Aquaporin 4 / Q000096:biosynthesis / Q000235:genetics; D001709:Biotechnology; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D050918:Phosphatidylethanolamine N-Methyltransferase / Q000096:biosynthesis / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C496030:AQP4 protein, human; D051401:Aquaporin 4; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C559160:PEMT protein, mouse; D050918:Phosphatidylethanolamine N-Methyltransferase