Supplementary Material
Polymer-based cell-free expression of ligand-binding family B G-protein coupled receptors
without detergents
Christian Klammt, Marilyn H. Perrin, Innokentiy Maslennikov, Ludovic Renault,
Martin Krupa, Witek Kwiatkowski, Henning Stahlberg, Wylie Vale and Senyon Choe
Includes:
Characterization of NVoy for membrane protein studies
Methods
References
Supplementary Figures S1, S2
Supplementary Table SI
Characterization of NVoy for membrane protein studies.
NVoy is a linear, uncharged molecule composed of a polyfructose (25mer) backbone
complemented with derivatized hydrophobic side chains with an overall molecular weight of 5
kDa which was introduced as an additive employed to increase solubility of hydrophobic
proteins. Our analysis by static light scattering (LS) coupled with refractive index (RI) and size
exclusion chromatography (SEC) revealed that this polymer forms stable micelle-like multimers
of approximately 112 kDa (22 polymer molecules) in aqueous solution. This value was
established using the polymer-specific constant [dn/dc]NVoy = 1.317 ml/g (the change of the
refractive index with concentration) determined as in1.
We found that NVoy can be used instead of detergents for IMP extraction from the
membrane and subsequent solubilization. Two different E. coli IMPs, a histidine kinase receptor
Etk and GFP-fused YcjF, were expressed in the E. coli membrane and extracted from membrane
fractions using different concentrations of NVoy. Fig. S1 shows membrane extraction for a range
of NVoy concentrations (1 to 5 mM). Quantities of extracted YcjF-GFP (Fig. S1A) and Etk (Fig.
S1C) were visualized by Coomassie-stained SDS-PAGE and, additionally, for YcjF-GFP, by the
intensities of in-gel GFP fluorescence (Fig. S1B). Both proteins were nearly completely extracted
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using NVoy at concentrations above 3 mM. TEV protease successfully cleaved YcjF-GFP in the
presence of 5 mM polymer as illustrated by the complete separation of the IMP and GFP (Fig.
S1A, B).
To test NVoy’s ability to replace detergents in protein-detergent complexes (PDCs) on
immobilized IMP samples, we used Etk extracted from the E. coli membrane by ndodecylphosphocholine (FC12) and YcjF-GFP extracted by n-dodecyl--D-maltoside (DDM).
Neither sample showed residual extraction detergent following detergent replacement by NVoy
during immobilized metal ion affinity chromatography (IMAC). As illustrated in Fig. S1D, the
completeness of this exchange for Etk was confirmed by the disappearance of the characteristic
FC12 signal as measured by 1D NMR in the elution sample. And in turn, we found that NVoy
can be completely exchanged with detergents in protein sample during IMAC as verified by
NMR measurements.
Methods
Cloning procedures and protein analysis.
GPCR targets were amplified from cDNA by standard polymerase chain reaction
techniques using Vent DNA-polymerase (New England Biolabs, MA, USA). For CCR5, an E.
coli codon optimized gene (Geneart AG, Germany) was used. Suitable restriction sites were
added to the DNA fragments with suitable oligonucleotide primers (Table S1). Purified PCR
fragments were inserted after cleavage into pET21a (Merck Biosciences, Germany) or pIVEX2.3
(Roche Applied Science, IN, USA) vectors. The resulting vector constructs contained an Nterminal T7 tag for pET21a derived constructs and a C-terminal His6 tag for both vector
derivatives.
The Invitrogen gel electrophoresis system (Invitrogen, CA, USA) was used for all SDSgel analyses following the manufactures protocol. NVoy extracted IMP samples supplemented
with SDS sample buffer were loaded on 4-20% (w/v) Novex® Tris-Glycine gels stained with
coomassie blue after GFP imaging, whereas GPCR samples were run on 12% NuPAGE® BisTris Gels using MES buffer and stained with coomassie blue or InstantBlue (Expedeon Protein
Solutions Ltd, UK).
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For western blot analysis, the gels were blotted on a 0.45 µm Immobilon-P
poly(vinylidene difluoride) membrane (Millipore, Germany) using Invitrogens Xcell IITM Blot
Module for 1 h at 35 volts. The membrane was then blocked for 1 h in blocking-buffer (1x Tris
buffered saline (TBS), 7% milk powder, 0.1% (w/v) Tween-20) and subsequently incubated for 1
hour with horseradish peroxidase (HRP)-conjugated T7-tag antibody (Merck Biosciences,
Germany) or HRP-conjugated His6-tag antibody (Abcam Inc., MA, USA) using 1 : 5000 and 1 :
1000 dilution in washing buffer (1x TBS, 0.1% Tween-20), respectively. After extensive washing
in washing buffer, the blots were analyzed by chemiluminescence (ECL western blot substrate,
Pierce, IL, USA) on X-Ray film (CL-XPosureTM, Pierce, IL, USA) using exposure times between
10 and 60 seconds.
Membrane preparation, extraction and TEV cleavage.
The YcjF-GFP construct, containing a TEV cleavage site between YcjF and GFP and a Cterminal His8 tag, was kindly provided by the von Heijne group. The Etk gene was cloned to a
Gateway-adapted pHis vector2 containing a N-terminal His9 tag. Both proteins were expressed in
E. coli BL21 DE3 cells (Invitrogen, CA, USA). Cells were grown overnight at 18°C after
induction by 0.5 mM IPTG at OD600 = 1, harvested, resuspended in lysis buffer (20 mM Tris-HCl
pH 8.0, 10 mM EDTA, 5 mM BME, 0.1 mM PMSF) and lysed with a M-100L CF microfluidizer
(Microfluidics, MA, USA). The pellet from a highspeed centrifugation (100,000 g, 1h) was
suspended in the lysis buffer and centrifuged at a low speed (10,000 g, 20 min) in order to
separate heavier inclusion bodies and other cell debris, followed by a second high speed
centrifugation. The crude membrane fraction was resuspended in salt wash buffer (20 mM TrisHCl pH 8.0, 0.5 M NaCl, 20% Glycerol, 5 mM BME, 0.1 mM PMSF) and frozen at -80°C.
Thawed membrane fractions of YcjF-GFP and Etk were divided into 5 times 100 µl
aliquots and mixed with 100 µl of 2x solubilization buffer (20 mM Tris-HCl pH 7.6, 300 mM
NaCl, 1 mM MgCl2) containing the NVoy polymer (NVoy = NV10, Expedeon Protein Solutions
Ltd, UK) at 5 different concentrations (10 mM, 8 mM, 6 mM, 4 mM and 2 mM), resulting in
final polymer concentrations of 5 mM, 4 mM, 3 mM, 2 mM and 1 mM for extraction. Samples
were stirred overnight (12 – 16 hours) at RT and subsequently centrifuged at 100,000 g for 1 hour
in order to separate polymer extracted protein in the supernatant from membrane fractions. The
supernatant of 5 mM NVoy extracted YcjF-GFP was cleaved by TEV protease using AcTEV
(Invitrogen, CA, USA) following the supplied protocol and incubated overnight (16 hours). For
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testing extraction properties of PMAL-B-100 (Calbiochem, NJ, USA) concentrations up to 0.44
mM were analyzed.
Detergent to Polymer exchange.
N-dodecyl--D-maltoside (DDM) (Anatrace, OH, USA) extracted YcjF-GFP and ndodecylphosphocholine (FC-12) (Anatrace, OH, USA) extracted Etk were analyzed for detergent
to polymer exchange using affinity chromatography. The exchange was tested with three polymer
concentrations using 3 Ni-ITA columns with 1 ml slurry equilibrated in wash buffer (20 mM
Tris-HCl pH 7.6, 300 mM NaCl, 1 mM MgCl2, 10 mM Imidazol) and 0.02 mM NVoy. 3 times
400 uL of YcjF-GFP (DDM) or Etk (FC-12) were loaded on 0.8 mM NVoy in wash buffer. Wash
fractions were collected for NMR analysis. 3 columns were washed in a second step with 3 times
3-column volumes of 0.02, 0.1, 0.2 mM NVoy, respectively, keeping the wash fractions for
subsequent NMR analysis. Protein was eluted with 3 times 1-column volume (0.5 ml) of elution
buffer (wash buffer containing 300 mM Imidazol) containing 0.02, 0.1, 0.2 mM NVoy,
respectively.
Detergent exchange to NVoy validation by NMR.
A Bruker Avance 700 MHz NMR spectrometer equipped with a cryogenic probe was
used for NMR analysis. To determine the real concentration of the components in the samples
they were mixed with the equal volumes (100 µl or 250 µl) of 1.0 mM 2,2-dimethyl-2silapentane-5-sulfonic acid (DSS) solution in D2O and complemented to 500 µl with D2O. NMR
spectra were acquired with 64 scans with 5 s relaxation delay and acquisition time of 2.7 s. The
signal of the (CH3)3-Si-group of DSS was used as the 1H chemical shift (0.0 ppm) reference.
Fractions of detergent exchange to polymer were analyzed by NMR tracking integrals of the
residual detergent signals as described previously3. In particular, the NVoy specific chemical
shifts at 4.24 ppm (corresponding to the protons of the fructose ring) were compared with the FC12 signal at 4.28 ppm (CH2-N+-(CH3)3).
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References for Supplementary Material
1.
Strop P, Brunger AT (2005) Refractive index-based determination of detergent concentration
and its application to the study of membrane proteins. Protein Sci 14:2207-2211.
2.
Kefala G, Kwiatkowski W, Esquivies L, Maslennikov I, Choe S (2007) Application of Mistic
to improving the expression and membrane integration of histidine kinase receptors from
Escherichia coli. J Struct Funct Genomics 8:167-172.
3.
Maslennikov I, Kefala G, Johnson C, Riek R, Choe S, Kwiatkowski W (2007) NMR
spectroscopic and analytical ultracentrifuge analysis of membrane protein detergent
complexes. BMC Struct Biol 7:74.
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Supplementary Figures
Figure S1
NVoy extracts IMPs from E. coli membrane fractions.
YcjF-GFP (A), (B) and Etk (C) were extracted by different NVoy concentrations (arrows) and
analyzed by Coomassie-stained gel and in-gel GFP fluorescence for YcjF-GFP (B). The
membrane fractions before pelleting the membranes (lane 1, 3, 5, 7, 9, 13) are compared to
extracted protein fractions (lane 2, 4, 6, 8, 10, 11, 14-18). TEV cleavage was performed at 5 mM
NVoy and cleaved products are indicated by arrows (lane 12). Polymer concentration and TEV
cleavage is indicated below the gels. (D) 1D-1H-NMR spectra of ETK samples exchanged from
FC-12 to NVoy. ETK, extracted in FC-12 and immobilized on Nickel beads was washed (W)
with 5 x 2 CV in 0.8 mM NVoy (W1-5), subsequently washed with 3 x 3 CV of 0.2 mM (W7)
before elution in buffer containing 0.2 mM NVoy (E1). The corresponding 1H chemical shifts of
FC12 and NVoy are indicated.
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Figure S2
NMR spectra of CRFR2 in detergent LMPG.
[1H,15N]-TROSY-HSQC spectra of ~50 µM U-15N-CRFR2, produced by P-CF and solubilized
in 2% LMPG (PL-CF), 20 mM HEPES-NaOH (pH 7.5) measured at 314K on a 700 MHz
spectrometer equipped with a cryogenic probe.
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Supplementary Tables
Table SI
Oligonucleotide primers for the construction of plasmids for CF expression of analyzed GPCRs.
* for CCR5, an E. coli codon optimized synthetic gene was used for cloning.
Construct
vector
primer Sequence (attached restriction linkers are underlined)
CCR1
pIVEX2.3
5’
CGG CC A TG GAA ACT CCA AAC ACC ACA GAG GAC TAT
3’
CGG CCC GGG GAA CCC AGC AGA GAG TTC ATG CTC CCC
5’
CGG CC A TG GAT TAT CAG GTG AGC AGC CCG ATT TAT G
3’
CGG CCC GGG CAG GCC AAC GCT AAT TTC CTG TTC GCC
5’
CGC GGA TCC ATG GAC ATG GCG GAT GAG CCA
3’
CCG CTC GAG AAT GGA GAC CTC CAA ACC AGT ATC
5’
CGC GGA TCC ATG GAG CCC CTG TTC CCA GCC
3’
CCG CTC GAG GGG CTT ATG CAG ACC AGC AAG CTG
5’
CGG CC A TG GGT TCC CTC CAG GAC CAG CAC TGC GAG AGC
3’
CGG GAG CTC G GAC TGC TGT GGA CTG CTT GAT GCT GTG
5’
CGG GAG CTC CAA CCA GGC CAG GCA CCC CAG GAC CAG
3’
CGG CTC GAG CAC AGC AGC TGT CTG CTT GAT GCT GTG
5’
CGG CC A TG GGT CAA CCA GGC CAG GCA CCC CAG GAC CAG
3’
CGG GAG CTC G CAC AGC AGC TGT CTG CTT GAT GCT GTG
5’
CGG GGA TCC GAA AAC GCC AGC ACA TCC CGA GGC TGT
3’
CGG CTC GAG CCA AAG GTG TCT TCC TGT GTG ACT TGG
5’
CGG GAG CTC ATG GCT ACA ACA GTC CCT GAT GGT TGC
3’
CGG CTC GAG GCT GCC CTC TTT CTT TAC TTC ATA GTC
CCR5*
SSR2
SSR5
CRFR1
CRFR2T7
CRFR2
GPRC5b
RAI3
pIVEX2.3
pET21a
pET21a
pIVEX2.3
pET21a
pIVEX2.3
pET21a
pET21a
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