Insert regions of yeast vectors used for expression of transmembrane proteins. Note that the indicated regions are not drawn to scale.

Expression of targeted yeast ORFs. Yeast strains expressing ORFS fused to C-terminal His6-ZZ tags from the MORF library [12]were cultured in YPD media until glucose depletion then induced with galactose (2%). After solubilization in 0.5% FC-16, cell lysates were subjected to small scale rapid affinity purification on IgG-Sepharose as described in Materials and Methods with elution by treatment with SDS-urea loading buffer. Each lane of the Coomassie-stained gel contained the protein derived from approximately 250 O.D._{600 ×} ml of culture, normalized based on wet pellet weight. The unfilled arrows indicated bands corresponding to expressed target proteins. The filled arrows at right indicate major bands eluted from the IgG Sepharose by SDS treatment even in the absence of loaded protein.

Purification of Bor1p, Ste24p. A)(top) Preparative Superdex 200 gel filtration chromatography during purification of Bor1p expressed from vector pSGP40 and cleaved from IgG Sepharose using GST-tagged 3C protease. The flow rate was 0.8 ml/min. (bottom) SDS polyacrylamide gel electrophoresis of fractions from the gel filtration separation. The molecular masses of marker proteins are indicated in kDa. B) SDS polyacrylamide gel electrophoresis of Bor1p following concentration of the purified protein. Lane 1) Molecular Weight markers; Lane 2) 10 μl purified protein; Lane 3) 5 μl purified protein; Lane 4) 5 μg purified GST-tagged 3C protease. C) Preparative Superdex 200 gel filtration chromatography during purification of Ste24p expressed from the MORF library vector [12] and cleaved from IgG Sepharose using GST-tagged 3C protease. The flow rate was 0.5 ml/min. (bottom) SDS polyacrylamide gel electrophoresis of fractions from the gel filtration separation. D) SDS polyacrylamide gel electrophoresis of Ste24p following concentration of the purified protein. Lane 1) 25 μl purified protein. Lane 2) 12.5 μl purified protein; Lane 3) 6.3 μl purified protein.

A) Inefficient cleavage from affinity matrix. Bor1p was expressed with a ZZ-His6 tag from the MORF vector BG1766 [12]. The membrane fraction from 300 O.D. ₆₀₀ × mls was solubilized in 1% DDM, then bound to IgG-Sepharose and eluted by cleavage overnight with the indicated amounts of GST-tagged 3C protease in the presence of 0.01% DDM. The eluted material was solubilized in SDS loading buffer, then the equivalent of 300 O.D.₆₀₀ × ml from each culture was separated by SDS polyacrylamide electrophoresis followed by staining with Coomassie blue. Lane 2 contains IgG-Sepharose to which no protein had been added, treated with SDS-urea loading buffer containing 2 mM Pefabloc SC. Lane 3 contains Bor1p eluted from IgG-Sepharose using SDS-urea loading buffer rather than 3C protease. Lanes 4 and 9 contain the indicated amounts of purified GST-tagged 3C protease. Lanes 8 and 13 show material that remained on the column after overnight elution with 3C protease but that could subsequently be eluted by treatment with SDS-urea loading buffer. B) Inefficient elution of cleaved protein from IgG. A mutated version of STE2 containing the N25Q and N32Q to prevent glycosylation [29] was expressed with a ZZ-His10 tag from vector pSGP40. The membrane fraction containing 400 μg total membrane protein was solubilized in 1% DDM, applied to 40 μl IgG-Sepharose, washed in 0.1% DDM, then eluted by overnight treatment with GST-tagged 3C protease (lane 6). Following elution with 3C, the material remaining associated with the IgG-Sepharose was eluted by sequential treatment with 5% SDS (lane 7), then SDS-urea loading buffer (lane 8). For reference, the same amount of membrane protein was bound to IgG-Sepharose and eluted directly with 5% SDS (lane 3), followed by SDS-urea buffer (lane 4). Lane 1 contains molecular weight markers, as indicated. Lane 2 contains 5 μg of purified GST-tagged 3C protease. Lane 5 shows a mock elution in which IgG-Sepharose to which no protein had been added was treated with SDS-urea loading buffer.

Elution profiles of purified preparations of 20 μg Bor1p using analytical size exclusion chromatography (TDSEC) on a Shodex KW-803 silica column. The protein used for each panel had been initially solubilized in 1% DDM. A) Static light scattering traces for the protein purified in DDM before and after 4 cycles of ∼10-fold dilution and reconcentration (in buffer containing 0.001% DDM) using a Millipore Amicon regenerated cellulose 50 kDa concentrators. The protein had initially been concentrated 9-fold after elution from the preparative size exclusion column in 0.05% DDM. B) TDSEC of Bor1p following cycles of dilution and reconcentration in DDM containing no added lipid. The protein had been exchanged into 0.1% DDM during affinity chromatography, then to 0.02% DDM during preparative size exclusion chromatography. C) TDSEC for Bor1p in C₁₂E₈ with lipid (phosphatidylserine (C18:0,C18:1), 0.1 mg/ml in a solution containing 6 mg/ml protein) added after the final cycles of protein dilution and re-concentration. The protein had been exchanged into 0.05% C₁₂E₈ during affinity chromatography, then to 0.03% C₁₂E₈ during preparative size exclusion chromatography. D) TDSEC of Bor1p in C₁₂E₈ with lipid (phosphatidylserine (C18:0,C18:1), 0.1 mg/ml; (at ∼0.6 mg/ml protein) added prior to the final cycles of dilution and re-concentration. The protein had been exchanged into 0.05% C₁₂E₈ during affinity chromatography, then to 0.03% C₁₂E₈ during preparative size exclusion chromatography.

Workflow for expression of yeast transmembrane proteins in yeast.

Sequences of insertion sites of expression vectors. A) Sequence of insertion region of vector pSGP36 expressing target proteins under galactose control fused to a cleavable C-terminal His10 tag. The thin arrows indicate the cleavage sites of BstXI. The thick arrow shows the site of insertion of assembled insert region into the PmlI site of BG1766 during creation of the vector. 5′ sequences from PGK1 are shown in bold. One base internal to the PGK1 sequences was altered to reduce secondary structure in the synthetic oligonucleotides during assembly. B) Sequence of pSGP36 and pSGP46 modified by insertion of a cloned reading frame. The open arrowhead shows the site of rhinovirus 3C cleavage. C) Sequence of insertion region of pSGP40 expressing proteins under galactose control fused to a C-terminal cleavable ZZ-His10 tag. The thick arrow shows the site of insertion of assembled insert region into the PmlI site of BG1766 during creation of the vector. D) Sequence of the pSGP38 and pSGP40 and pSGP46 following insertion of a reading frame using LIC cloning. The open arrowhead shows the site of rhinovirus 3C cleavage.

Reduction of activity of endogenous yeast proteases by PMSF and use of protease-deleted strains. A) Inhibition of proteolytic activity in purification of Ste24p. Membranes were prepared from 300 O.D._{600 ×} ml of cultures of the indicated yeast strains expressing Ste24p with a ZZ-His10 tag (vector pSGP40) that had been lysed in the presence 1 mM AEBSF. The membranes were solubilized in 0.5% FC-16 in the presence (lanes 2,4,and 6) or absence (lanes 3,5, and 7) of 170 μg/ml PMSF, bound to 40 μl Talon, then eluted using 5 μg of GST-tagged 3C protease. B) PMSF inhibition of proteolysis in purification of Ste24p. A total of 300 O.D._{600 ×} ml of lysate from yeast strain S258C expressing Ste24p with a ZZ-His10 tag (vector pSGP40), prepared in the presence of 1 mM AEBSF, 2.5 μg/ml leupeptin, and 2.5 μg/ml pepstatin was directly solubilized in 0.5% FC-16 in the absence of protease inhibitors (without preparing a membrane fraction), then bound to 20 μl of IgG-sepharose. The IgG-sepharose was washed in 0.1% FC-16 in the absence (lanes 3-6) or presence (lanes 9-12) of PMSF, then the protein was eluted by treatment with 25μg GST-tagged 3C protease. Following cleavage (elution 1, lanes 3 and 9)), the IgG-sepharose was washed twice with buffer containing 0.1% FC-16 (elutions 2 and 3; lanes 4, 5, 10, and 11). Material remaining on the column following treatment with 3C protease was then eluted by treatment with SDS-urea loading buffer containing (lanes 6 and 12). For reference, the figure also shows the results of SDS-urea treatment of IgG-sepharose onto which no protein had been loaded (lanes 2 and 13). Samples containing 25 μg of purified GST-tagged 3C protease are shown in lanes 7 and 14 for reference.

Effects of imidazole on IMAC binding. Membranes from 800 O.D.₆₀₀ × ml of cells expressing Ste2p tagged with ZZ-His10 (expressed from vector pSGP46) were initially solubilized in 1% FC-16. The expressed allele of STE2 contained the substitutions N25Q and N32Q to prevent glycosylation [29]. The solubilized membranes were applied to 30 μl Talon® in 50 mM Hepes, pH 7.5, 150 mM NaCl, 15% glycerol, 2.5 μg/ml pepstatin A, 1 μg/ml chymostatin, 2.5 μg/ml leupeptin, 1 mM AEBSF, and 2 mM β-mercaptoethanol) in the presence of the indicated concentrations of imidazole, then washed four times (800 μl each time) in buffer that contained the same imidazole concentration but a reduced concentration of FC -16 (0.1%) and only 1 μg/ml chymostatin for protease inhibition. Protein was eluted using 100 μl of similar buffer containing 300 mM imidazole, then concentrated using Strataclean™ (Agilent Technologies). The entire eluate was loaded on the gel, which was stained with Coomassie Blue.

Decrease in levels of DDM in the concentrator concentrate during repeated dilutions and re-concentration of Bor1p. A purified preparation of Bor1p was subjected to repeated cycles of 10-fold dilution with size exclusion buffer containing 0.001% DDM, followed by re-concentration using an Amicon Ultra (50 kDa cut-off) to the original volume. Detergent concentrations were determined by colorimetric assay [27]. The total amount of detergent is shown for a sample in which the volume changes at different steps of concentration. The final concentrations of protein and detergent were 10 mg/ml and 1.8 % w/v in a final volume of 300 μl.