Flowchart illustrating the screening process for the overexpression and purification of eukaryotic membrane protein-GFP fusions in Saccharomyces cerevisiae.

Cloning by homologous recombination into 2 μ Saccharomyces cerevisiae GFP-fusion vector. (a) The amount of PCR and vector used in cloning (as stated in Box 1) is depicted in the UV-exposed agarose-gel inset: i, SmaI linearilized vector; ii, amplified gene with overhangs required for homologous recombination. (b) Cloning site used in our 2μ GFP-fusion vector pDDGFP-2 (ref. 6). TEV, tobacco etch virus; yEGFP, yeast-enhanced green fluorescent protein.

Estimating reliable expression levels of eukaryotic membrane protein-GFP fusions from S. cerevisiae whole-cell fluorescent measurements. (a) In-gel fluorescence of crude yeast membrane samples isolated after the overexpression of 20 different eukaryotic transport proteins (Steps 6–18): arrowhead, membrane protein-GFP fusion; asterisk, endogenous fluorescent ‘background’ protein. (b) Comparison of whole-cell GFP counts (Steps 4–11) for 15 yeast and mammalian membrane protein-GFP fusions grown in either a 2.5-l shaker flask (Steps 27–29) or a 15-/50-l fermenter. (c) Whole-cell GFP counts from 10-ml culture volume for 10 yeast and 24 mammalian membrane proteins versus their GFP counts from membranes isolated from 10-l cultures; membrane pellet was resuspended in 6 ml of membrane resuspension buffer (MRB) per 1 l of initial cell culture volume (Steps 27–32). (d) Calculated membrane protein overexpression levels (as outlined in Box 2 with the exception that Step 7 was omitted) in whole-cells versus membranes for fusions presented in panel c. Red square, data for a membrane protein-GFP fusion of ‘typical’ recovery, which is analyzed further in panel e. (e) In-gel fluorescent SDS-PAGE gel for fusion depicted as red square in panel d: left lane, 10 μl of membranes after resuspension into prespin volume; middle lane, 10 μl of high-speed spin supernatant; right lane, pure GFP standard. Using quantification software band intensities were measured. RFU, relative fluorescence units.

Examples of membrane protein GFP-fusion localization in S. cerevisiae as monitored by confocal microscopy. Top, plasma membrane protein Ctr1p; top-middle, Golgi membrane protein Hut1p; middle-bottom, Endoplasmic reticulum (ER) membrane protein Isc1p; bottom, vacuolar membrane protein Uga4p (examples have been published in core ref. 6).

Flowchart illustrating the purification of eukaryotic membrane proteins from GFP-fusions. (a) Detergent solubilization of membrane protein- GFP fusion (Step 39). (b) Incubation of supernatant with Ni-NTA resin (Step 42). (c) Immobilized metal-affinity chromatography (IMAC) purification (Steps 43–45). (d) Dialysis and cleavage of GFP from membrane protein-GFP fusion (Step 47). (e) Reverse IMAC to remove GFP and tobacco etch virus (TEV) from membrane protein (Steps 49 and 50). (f) Gel filtration of membrane protein (Step 51): example shown here is for Homo sapiens sugar transporter with the pooled fraction analyzed by SDS-PAGE (Step 53) as shown in inset.

Determining the monodispersity of membrane protein-GFP fusions in different detergents using fluorescence size-exclusion chromatography (FSEC). (a) FSEC traces showing the effect of cholesterol hemisuccinate salt addition on the detergent extraction efficiency and monodispersity of a Homo sapiens metal transporter. (b) FSEC traces of membranes containing Rattus norvegicus ABC transporter-GFP fusion after adding C₁₂E₉: before ultracentrifugation, dark blue line; after ultracentrifugation, light-blue line. (c) FSEC traces obtained for a yeast trafficking chaperone protein in the five recommended initial screening detergents (Steps 33–38). Aggregated protein in void peak, membrane protein-GFP fusion and ‘free’ GFP peaks are as highlighted. Inset (top), the SEC trace for the n-dodecyl-β-D-maltopyranoside (DDM)-solubilized total membranes of the sample shown in panel c, main. Inset (bottom), the SEC trace of yeast chaperone purified in N,Ndimethyldodecylamine N-oxide (LDAO) using Steps 36–51. CHS, cholesterol hemisuccinate Tris salt; DM, n-Decyl-β-D-maltopyranoside; RFU, relative fluorescence units.

Examples of purified eukaryotic transport proteins. (a) SDS-PAGE gel as analyzed by in-gel fluorescence and Coomassie staining for the purification of a Homo sapiens sugar transporter: 1, prestained molecular weight (MW) protein ladder; 2, total membranes; 3, n-dodecyl-β-D-maltopyranoside (DDM)-solubilized membranes before ultracentrifugation; 4, DDM-solubilized membranes after ultracentrifugation; 5, Ni-NTA flowthrough; 6, Ni-NTA elution; 7, elution of reverse Ni-NTA after GFP cleavage; 8, flowthrough of reverse Ni-NTA after GFP cleavage; 9, purified membrane protein after SEC; 10, fluorescent MW protein ladder; 11, pure GFP standard. (b) Assay for the binding of a specific inhibitor (nanomole affinity) to a Rattus norvegicus amino acid transporter, left lane, buffer solution; middle lane, GFP; right lane, amino acid transporter-GFP fusion.