PMID- 21925269 OWN - NLM STAT- MEDLINE DCOM- 20120524 LR - 20211020 IS - 1095-9130 (Electronic) IS - 1046-2023 (Print) IS - 1046-2023 (Linking) VI - 55 IP - 4 DP - 2011 Dec TI - Efficient expression screening of human membrane proteins in transiently transfected Human Embryonic Kidney 293S cells. PG - 273-80 LID - 10.1016/j.ymeth.2011.08.018 [doi] AB - It is often an immense challenge to overexpress human membrane proteins at levels sufficient for structural studies. The use of Human Embryonic Kidney 293 (HEK 293) cells to express full-length human membrane proteins is becoming increasingly common, since these cells provide a near-native protein folding and lipid environment. Nevertheless, the labor intensiveness and low yields of HEK 293 cells and other mammalian cell expression systems necessitate the screening for suitable expression as early as possible. Here we present our methodology used to generate constructs of human membrane proteins and to rapidly assess their suitability for overexpression using transiently transfected, glycosylation-deficient GnT I-HEK 293 cells (HEK 293S). Constructs, in the presence or absence of a C-terminal enhanced green fluorescence protein (EGFP) molecule, are made in a modular manner, allowing for the rapid generation of several combinations of fusion tags and gene paralogues/orthologues. Solubilization of HEK 293S cells, using a range of detergents, followed by Western blotting is performed to assess relative expression levels and to detect possible degradation products. Fluorescence-detection size exclusion chromatography (FSEC) is employed to assess expression levels and overall homogeneity of the membrane proteins, to rank different constructs for further downstream expression trials. Constructs identified as having high expression are instantly suitable for further downstream large scale transient expression trials and stable cell line generation. The method described is accessible to all laboratory scales and can be completed in approximately 3 weeks. CI - Copyright (c) 2011. Published by Elsevier Inc. FAU - Chaudhary, Sarika AU - Chaudhary S AD - Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, United States. FAU - Pak, John E AU - Pak JE FAU - Pedersen, Bjorn P AU - Pedersen BP FAU - Bang, Lois J AU - Bang LJ FAU - Zhang, Liye B AU - Zhang LB FAU - Ngaw, Samantha M M AU - Ngaw SM FAU - Green, Raissa G AU - Green RG FAU - Sharma, Vinay AU - Sharma V FAU - Stroud, Robert M AU - Stroud RM LA - eng GR - R37 GM024485/GM/NIGMS NIH HHS/United States GR - R01 GM024485/GM/NIGMS NIH HHS/United States GR - U54 GM094625/GM/NIGMS NIH HHS/United States GR - P50 GM73210/GM/NIGMS NIH HHS/United States GR - R37 GM24485/GM/NIGMS NIH HHS/United States GR - P50 GM073210/GM/NIGMS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20110908 PL - United States TA - Methods JT - Methods (San Diego, Calif.) JID - 9426302 RN - 0 (Membrane Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 0 (enhanced green fluorescent protein) RN - 147336-22-9 (Green Fluorescent Proteins) SB - IM MH - Blotting, Western MH - Chromatography, Gel MH - Cloning, Molecular MH - Gene Expression MH - Genetic Vectors MH - Green Fluorescent Proteins/biosynthesis/genetics/isolation & purification MH - HEK293 Cells MH - Humans MH - Membrane Proteins/*biosynthesis/genetics/isolation & purification MH - Polymerase Chain Reaction MH - Recombinant Fusion Proteins/*biosynthesis/genetics/isolation & purification MH - Solubility MH - Transfection PMC - PMC3600976 MID - NIHMS430465 EDAT- 2011/09/20 06:00 MHDA- 2012/05/25 06:00 PMCR- 2013/03/18 CRDT- 2011/09/20 06:00 PHST- 2011/06/15 00:00 [received] PHST- 2011/08/27 00:00 [revised] PHST- 2011/08/31 00:00 [accepted] PHST- 2011/09/20 06:00 [entrez] PHST- 2011/09/20 06:00 [pubmed] PHST- 2012/05/25 06:00 [medline] PHST- 2013/03/18 00:00 [pmc-release] AID - S1046-2023(11)00163-0 [pii] AID - 10.1016/j.ymeth.2011.08.018 [doi] PST - ppublish SO - Methods. 2011 Dec;55(4):273-80. doi: 10.1016/j.ymeth.2011.08.018. Epub 2011 Sep 8.