Yeast strains
Production of tagged protein constructs for purification was carried out in
S. cerevisiae strain PAP1500 (α
ura3-52 trp1::GAL10-GAL4 lys2-801 leu2
Δ1 his3 Δ
200 pep4::HIS3 prb1
Δ
1.6R can1 GAL) [30] while complementation studies were
performed in PAP7111 (α
ura3-52 his3
Δ
200 HIS 4-15 trk1
Δ trk2 Δ
::HIS3 PMA1::mcherry). PAP7111 was
constructed by transformation of CY162 [50] with a PCR fragment carrying the mCherry [51] coding region flanked by 35 nucleotides
used for homologous recombination with the chromosomal PMA1 locus. The wild type strain BY4741 (a his3
Δ
1 leu2
Δ
0 met15
Δ
0 ura3
Δ
0) [52] was used in the complementation studies, too.
Recombinant plasmid construction
A 3480 bp long yeast codon optimized hERG sequence was purchased from
Genscript, USA. To C-terminally tag hERG with a Tobacco Etch Virus cleavage site
and a yEGFP-His 8 sequence, we PCR amplified codon
optimized hERG cDNA with primers hERGfw 5 ′-ACACAAATACACACACTAAATTACCGGATCAAT
TCTTTAAAACGAATGCCAGTTAGAAGAGG TC-3 ′ and hERGrv 5
′-AAAT
TGACTTTGAAAATACAAATTTTC
ACTACCTGGGTCACTACCG - 3
′ and yEGFP cDNA [32] with primers GFPfwTEV 5
′-AAAATTTGTATTTTCAAAGTCAATTT
TCTAAAGGTGAAGAATTATTCACT-3
′ and GFPHISdo 5
′-CTT
CAATGCTATCATTTCCTTTGATATTGGATCATCTAATGGTGATG
GTGATGGTGATGGTGTTTGTACAATTCA-3
′.
The emphasized nucleotides were used for in
vivo homologous recombination, the bold nucleotides are identical
or inverse complimentary to the template, the text in between these two
formattings in hERGfw is the Kozak sequence from the yeast PMR1 gene and in the GFPHISdo it is the His-tag.
The TEV site is marked in italics. All PCR
reactions were performed with AccuPol DNA polymerase (Amplicon, Denmark). The
hERG-TEV-GFP-His 8 expression plasmid was generated by
in vivo homologous recombination by
transforming PAP1500 with hERG and GFP PCR products and SalI, HindIII and BamHI digested pEMBLyex4 [53] expression vector, using the
transformation protocol described by Gietz and Schiestl [54]. The correct nucleotide sequence of the
expression construct was verified by DNA sequencing at Eurofins MWG Operon,
Germany.
Yeast complementation assay
PAP7111 cells harboring the pEMBLYex4 plasmid, the hERG-TEV-GFP-His
8 expression plasmid or the hERG-His
10 expression plasmid were cultured in SD medium
[32] supplemented with 100 mM
KCl. The wild type strain BY4741 was cultured in SD medium supplemented with
histidine, leucine, methionine and uracil. Cells were subsequently harvested,
washed thoroughly with 18 m Ω H
2O to remove residual KCl originating from the
initial growth medium and inoculated in TES-TRIS buffered (pH 6.0) SD+SG medium
at OD 450=0.05 in 96 well micro plates (Nunc, clear
plastic) at KCl concentrations of 0, 0.1, 1, 2, 5, 10, 15, 20, 25, 30, 35 or 100
mM, respectively. Growth was monitored 3 times a day for 5 days by measuring OD
450.
Recombinant hERG production
Yeast cells were cultured and induced to express hERG as described by
Scharff-Poulsen, P and Pedersen, PA [32] In brief cells were inoculated in 5 ml synthetic minimal
(SD) medium supplemented with leucine and incubated at 30°C O/N until
saturation. The plasmid copy number in the yeast population was subsequently
increased by growth in medium lacking leucine. This culture was used to
inoculate 1 L of expression medium, which is SD medium with glucose (0.5% w/v),
glycerol (3% v/v), alanine (20 mg/L), arginine (20 mg/L), aspartic acid (100
mg/L), cysteine (20 mg/L), glutamic acid (100 mg/L), histidine (20 mg/L), lysine
(30 mg/L), methionine (20 mg/L), phenylalanine (50 mg/L), proline (20 mg/L),
serine (375 mg/L), threonine (200 mg/L), tryptophane (20 mg/L), tyrosine (30
mg/L) and valine (150 mg/L) to an OD 450 of 0.05. The
culture was incubated at room temperature until the OD
450 reached 1.0, transferred to 15°C and supplemented
with induction medium (identical to the expression medium described above except
that 20% galactose has substituted 0.5% glucose) to a final concentration of 2%
galactose. The culture was incubated for at least 48 hours before
harvesting.
Live cell bioimaging
Localization of heterologously expressed GFP-tagged hERG was determined by
visualizing GFP fluorescence in whole cells at 1000 × magnification, using a
Nikon Eclipse E600 microscope coupled to an Optronics Magnafire model S99802
camera.
Deglycosylation
80 μg of crude membranes were incubated
with 500 units of Endo-H (New Biolabs, USA) at 4°C in Lysis buffer over night
alongside 80 μg of crude membranes in
lysisbuffer with no added Endo-H. Samples were separated in a 10% SDS-PAGE gel
at 150 V for 2 hours, and visualized by in-gel fluorescence.
Temperature optimization of hERG production
Yeast cells were grown at room temperature as described above in 1 L of
expression medium. At OD 450=1.0, half of the culture was
transferred to 15°C and the other to 30°C. After thermo equilibration, hERG
production was induced by adding 55 ml of induction medium. Samples were
collected 12, 24, 48, 72 and 96 hours post induction. Crude membranes were
isolated from cells harvested at each time point and analysed by in-gel
fluorescence using a LAS 4000 (GE Healthcare, USA).
Membrane preparation
Crude yeast membranes were prepared by disrupting cell pellets by glassbead
vortexing [55]. Briefly, cell
pellets from 1 L cultures were resuspended in 10 ml ice cold lysis buffer (25 mM
imidazole, 1 mM EDTA, 1 mM EGTA, 10% glycerol (v/v) pH 7.5) with protease
inhibitors (1 mM PMSF, 1 mM benzamidine, leupeptin (1 μg/ml), pepstatin (1 μg/ml),
chymostatin (1 μg/ml) and aprotinin (1
μg/ml)). Samples were vortexed 4 × 1
minutes with at least 1 minute of cooling in between mixing. The liquid phase
was collected, and beads were washed several times with lysis buffer generating
samples of 50 ml total volume. Cell debris was pelleted by centrifugation for 10
minutes at 3,000 rpm and 4°C in an SS-34 rotor. Crude membranes were pelleted
from the supernatant by ultra-centrifugation for 1.5 hour at 40,000 rpm and 4°C
in a 70TI rotor. Crude membranes were resuspended in 3 ml lysis buffer with
protease inhibitors (as above), homogenized in a Potter-Elvehjem homogenizer and
stored at -80°C for further use.
Protein and hERG-GFP quantification
Protein concentrations in crude membranes were determined by the BCA assay
[56] according to the
manufacturer ′s specifications (Sigma, USA) using BSA
as a standard. The density of hERG-TEV-GFP-His 8 in yeast
membranes was determined from the GFP fluorescence emitted from 25 μg of total membrane protein measured in 96 well
white microplates (Nucleon Nunc) after adjustment of the volume to 200 μl with buffer (20 mM phosphate pH 7.0, 200 mM
NaCl, 10% glycerol, 10 mM Imidazole). Fluorescence was measured in a
spectrofluorometer (Fluoroskan Ascent, Thermo Scientific) using buffer as a
blank. Excitation was at 485 nm and emission at 520 nm. Fluorescence was
converted to pmol hERG-GFP from a standard curve generated from purified GFP
mixed with yeast membranes as previously established [31,32].
Astemizole binding to crude membranes
Crude membranes were used to assess the capability of the recombinant hERG-GFP
to bind the hERG ligand Astemizole, as described for HEK293 cells expressing
hERG [38]. Aliquots of 200
μg crude membrane protein in total
volumes of 400 μl incubation buffer (10 mM
HEPES 130 mM NaCl, 60 mM KCl 0.8 mM MgC l2 10 mM glucose
1 mM EGTA pH 7.4) supplemented with protease inhibitors (1 mM PMSF, 1 mM
Benzamidine, 1 μg/ml Leupeptin, Chymostatin,
Pepstatin and Aprotinin) were mixed with [
3H]-Astemizole concentrations ranging from 0.5 - 20
nM. Unspecific binding was determined in the presence of 10 μM non-radiolabeled Astemizole. Binding was done at
15°C for 2 hours with slow speed shaking, and samples kept on ice here on after.
Protein-ligand complexes were separated from free ligand by filtration of 200
μl sample through Whatman GF/B glass
fiber filters presoaked in 0.3% polyethyleneimine and washed once in 1 ml ice
cold wash buffer (25 mM Tris-HCl, 130 mM NaCl, 5 mM KCl, 0.8 mM MgC
l2, 0.05 mM CaCl 2, pH 7.4)
with protease inhibitors. Subsequently filters were washed 6 times with 1 ml ice
cold wash buffer using vacuum filtration, and bound ligand was detected using a
Perkin Elmer Tri-Carb 2910 TR liquid scintillation counter. 50 μl of unfiltered sample was used for determination
of total CPM counts. A subsequent BCA protein determination assay was done on
all samples to allow for corrections due to any protein loss during the binding
assay. After calculating total, unspecific and specific binding the resulting
graph was analyzed using the Sigmaplot non-linear regression tool, ligand
binding; one-site saturation (f = Bmax*abs(x)/(Kd + abs(x)) to estimate binding
affinity and capacity.
Astemizole binding to purified protein
0.1 μg of affinity purified
hERG-TEV-GFP-His 8 protein was used to estimate binding
affinity and binding capacity. Purified hERG protein was incubated with
increasing ammounts of [ 3H]-Astemizole, filtered and
[ 3H]-Astemizole binding determined by scintillation
counting. Unspecific binding was determined in presence of 10 μM non-radioactive labelled Astemizole. All
solutions contained 1.5 mg/ml FC-12 and 0.5 mg/ml CHS. Experimental data were
analysed in Sigmaplot using a sigmoid 3 parameter curve-fit (f=a(1+exp(-(x-x
0)/b).
Detergent screening
Crude membranes were incubated in buffer B (25 mM Tris-HCl, 10 mM Imidazole,
0.5 M NaCl, 10% glycerol, pH 7.6) supplemented with protease inhibitors (1 mM
PMSF, 1 mM Benzamidine and 1 μg/ml Leupeptin,
Chymostatin, Pepstatin and Aprotinin respectively) at protein:detergent:CHS
ratios (w/w) of 1:2:0.7; 1:3:1 or 1:4:1.4 The screen included detergents FC-12,
n-dodecylphosphocholine; LDAO, Lauryldimethylamine N-oxide; Cymal-5,
5-cyclohexyl-1-pentyl- β-D-maltoside; DDM,
n-dodecyl- β-D-maltopyranoside; DM, n-decyl-
β-Dmaltopyranoside; C
12
E
8, Octaethylene glycol monododecyl ether; CHAPS,
3-[(3chol-amidopropyl)-dimethylammonio]1-propane sulfonate/
N,N-dimethyl-3-sulfo-N-[3-[[3a,5b,7a,12a)-3,7,12- tri -
hydroxy-24-oxocholan-24-yl]amino]propyl]-1-propana- miniumhydroxide and Octyl
glucoside. All detergents were of Anagrade quality and purchased from
Affymetrix, UK. Solubilization was performed at slow rotation at 4°C for 1 hour.
Solubilized hERG-GFP channel protein was separated from un-solubilized cell
debris by ultra-centrifugation at 70,000 rpm for 30 minutes at 4°C in a Beckman
Optima™TLX ultracentrifuge fitted with an S.N. 96U 826 rotor. Fluorescence was
detected in microplates in a spectrofluorometer (Fluoroskan Ascent, Thermo
Scientific) using buffer as a blank. Excitation was at 485 nm and emission at
520 nm. Solubilization efficiency was etimated as fluorescence in the
supernatant divided by fluorescence in the crude membranes used for
solubilization.
FSEC
Solubilized crude membranes were analyzed by fluorescence size exclusion
chromatography (FSEC) on a Superose 6 10/300 column attached to an
\(\ddot {A}\)KTA Purifier (GE Healthcare, USA), using FSEC buffer (20 mM
TRIS-HCl, 0.15 M NaCl, 0.03% DDM). 1 μM
Astemizole was added to the buffer in experiments involving Astemizole. The
effluent from the Superose 6 10/300 column was coupled to a fluorescence
detector (Shimadzu Prominence RF-20A), to measure fluorescence and visualize the
elution profile of the GFP tagged hERG channel. To estimate the molecular weight
of the solubilized hERG-TEV-GFP-His 8 protein, we used
the HMW calibration kit from GE Healthcare dissolved at 20 mg/ml in FSEC buffer.
The molecular masses were: Ovalbumin 43 kDa; Conalbumin 75 kDa; Aldolase 158
kDa; Ferritin 440 kDa; Thyroglobulin 669 kDA; Blue Dextran 2000 kDa. The elution
volume for Blue Dextran defined the void volume.
Ni-NTA affinity purification
For purification, the hERG-GFP protein was solubilized in buffer B at a
protein:FC-12:CHS ratio of 1:3:1 (w/w/w) at slow rotation at 4°C for 1 hour.
Non-solubilized material was pelleted at 70,000 rpm in the Beckmann Optima TL200
ultracentrifuge for 30 minutes at 4°C. Solubilized membranes were diluted in
buffer B with protease inhibitors to a detergent concentration of 0.75 mg/ml
corresponding to 1.5 times CMC for Fos-choline-12 and a CHS concentration of
0.26 mg/ml, incubated over night with 1 ml of Ni-NTA Agarose (Qiagen, Germany)
at 4°C with slow magnetic stirring. The Agarose slurry was subsequently loaded
onto a 2 ml CellThru disposable column (Clontech, USA). After collection of the
run through, the column was washed with Buffer B containing 10 mM, 30 mM, 100
mM, 250 mM or 500 mM imidazole. All buffers contained 0.75 mg/ml FC-12 and 0.26
mg/ml CHS Fluorescence in each fraction was quantified using a
spectrofluorometer (Fluoroskan Ascent, Thermo Scientific) using buffer as a
blank. Excitation was at 485 nm and emission at 520 nm.
TEV cleavage
Purified hERG-GFP-His 8 fusion protein were digested
O/N in snakeskin dialysis bags (Thermo Scientific, USA) with dialysis buffer (20
mM phosphate pH 7.0 200 mM NaCl 0.075% (w/v) FC-12 0.026% (w/v) CHS) and TEV
protease [32] at room temperature
with a protein to TEV ratio of 1:10 (w/w). Digestion efficiency was estimated by
in-gel fluorescence followed by Coomassie staining.