title: Efficient expression screening of human membrane proteins in transiently transfected Human Embryonic Kidney 293S cells.
abstract: 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.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, United States.
authors.forename: Sarika
authors.initials: S
authors.lastname: Chaudhary
authors.forename: John E
authors.initials: JE
authors.lastname: Pak
authors.forename: Bjørn P
authors.initials: BP
authors.lastname: Pedersen
authors.forename: Lois J
authors.initials: LJ
authors.lastname: Bang
authors.forename: Liye B
authors.initials: LB
authors.lastname: Zhang
authors.forename: Samantha M M
authors.initials: SM
authors.lastname: Ngaw
authors.forename: Raissa G
authors.initials: RG
authors.lastname: Green
authors.forename: Vinay
authors.initials: V
authors.lastname: Sharma
authors.forename: Robert M
authors.initials: RM
authors.lastname: Stroud
mesh_terms: D015153:Blotting, Western; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D014162:Transfection
chemical_list: D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins