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Cell-free expression of integral membrane proteins
Christian Klammt, Innokentiy Maslennikov,
Witek Kwiatkowski and Senyon Choe
The Salk Institute, Structural Biology Laboratory, La Jolla, CA
Center for Structures of Membrane Proteins © 2006
Outline
Cell-free (CF) expression systems
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Overview (extract sources, reaction set-ups, commercial systems)
Protocols for set-up of an E. coli based CF expression system
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S30 cell extract preparation
T7-RNA polymerase preparation
List of reaction components
Stock solutions of CF components
Pipetting protocol
Reaction compartments for dialysis mode
CF expression of integral membrane proteins (IMPs)
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Possible modes for CF IMP expression
• P-CF (insoluble precipitate)
• D-CF (detergent)
• L-CF (lipids)
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List of detergents/ lipids used with CF system
Trouble shooting
N-CF – a new mode for CF expression of IMPs
Center for Structures of Membrane Proteins © 2006
CF expression as alternative for difficult proteins
Apoproteins
Disulfide
bonded
proteins
+ oxidative
cond., PDIs
+ modified aa
Modified
proteins
CF
Membrane proteins
Specifically
labeled
proteins
Labeled proteins
Modified proteins
Unstable proteins
+ detergents,
lipids
Membrane
proteins
Cytotoxic proteins
Metallo proteins
Disulfide bonded
proteins
Metallo
proteins
Unstable
proteins
Cytotoxic
proteins
Center for Structures of Membrane Proteins © 2006
Apo-proteins
CF extract sources and systems
Wheat germ
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low nuclease activities, long life-times (days), preparative system
difficult extract preparation, complex system
E. coli
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high efficiency, easy extract preparation, optimized protocols for
various applications available, preparative system
high variations, shorter life-times (hours)
Rabbit reticulocyte
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eukaryotic system
difficult extract preparation, complex non preparative system
Insect cells
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eukaryotic system, post-translational modifications
complex non preparative system
Center for Structures of Membrane Proteins © 2006
Reaction set-up - batch vs. dialysis
batch
Ribosomes,
ARSases,
tRNA,
Polymerases,
DNA, mRNA,
IFs, EFs, RFs,
NTPs,
amino acids,
Energy system
COUPLED
TRANSCRIPTION/
TRANSLATION
stirrer
SUBSTRATES:
amino acids,
NTPs, energy
LMW
substrates
PRODUCTS:
NMPs, PPi, Pi
Reaction mixture
(RM)
Dialysis membrane
Feeding mixture
(FM)
dialysis
Easy set-up
Need for suitable dialysis set-up
Short reaction times (<3 hours)
Long reaction times (> 12 hours)
Agglomeration of harmful LMW
products, fast energy
consumption
Continuous exchange of harmful
LMW products and supply of
energy
High throughput screening
Up to 6 mg/ml of protein in RM
Up to 1 mg/ml
Limited high throughput screening
Center for Structures of Membrane Proteins © 2006
Commercially available systems
E. coli:
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ExpresswayTM (Invitrogen)
RTS E. coli (Roche)
EasyExpress E.coli (Qiagen)
S30 T7 High-Yield (Promega)
Wheat germ
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ENDEXT® (Cell-Free Sciences)
RTS wheat germ (Roche)
TNT® wheat germ (Promega)
Rabbit reticulocyte
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TNT® rabbit reticulocyte (Promega)
Insect
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EasyExpress insect (Qiagen)
TNT® insect (Promega)
Center for Structures of Membrane Proteins © 2006
Roche is using a dialysis set-up
(patent), other systems are
based on batch mode.
Convenient, easy to use kits
High costs compared to
individual systems
“Black box” systems with
limited room for optimizations
E. coli based coupled transcription-translation system
Bacterial S30 extract
T7-RNA polymerase
Template DNA (T7-regulatory
sequences), e.g. pET-vectors
tRNA
Amino acids
RNase, protease inhibitors
Energy system: acetyl phosphate
posphoenolpyruvate, kinases
NTPs
Suitable buffer system
Mg2+-, K+-Ions
Additives: PEG, spermidine, cAMP,
oxalate, coenzyme A, NAD,…
Target specific stabilizers:
Detergents, lipids, chaperones,
cofactors, ligands, inhibitors,…
Center for Structures of Membrane Proteins © 2006
Coupled transcription/translation
E. coli S30 extract preparation (3 days)
Cell growth (day 1):
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Inoculate 5 liters of TB media at a ratio of 1:100 with fresh overnight culture of E. coli strain (A19, BL21, D10)
in a fermentor and start rapid cooling when cells reach log-phase (OD600 = 5-6). All following steps on ice!
Cell wash:
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Harvest cells by centrifugation (7,000 x g, 4°C, 10 min) and resuspend in Buffer A
(10 mM Tris-Acetat, pH 8.2, 14 mM Mg(oAc)2, 0.6 mM KCl, 6 mM BME) using a glass rod.
Centrifuge cells (8,000 x g, 4°C, 30 min) and repeat washing, [STORE ON ICE OVER NIGHT], wash 3rd time.
Cell lysis (day 2):
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Weight cells and resuspend in 110% w/v buffer B (10 mM Tris-Acetat, pH 8.2, 14 mM Mg(oAc)2,
0.6 mM KCl, 1mM DTT, 0.1 mM PMSF). Disrupt cells by French press (20,000 psi) or cell disrupter at 4°C.
Isolation of cell extract:
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Centrifuge disrupted cells (30,000 x g, 4°C, 30 min), keep first 2/3 of supernatant and centrifuge again.
keep 2/3 of second supernatant.
Run-off procedure:
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Supplement extract with 400 mM NaCl (5 M stock) and incubate for 45 min at 42°C.
Dialysis:
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Dialyse against 2x 40 volumes over night with first buffer exchange after 2 h into S30 buffer (10 mM TrisAcetat, pH 8.2, 14 mM Mg(oAc)2, 0.6 mM KoAc, 0.5 mM DTT at 4°C (25 kDa MWCO membrane).
Store extract (day 3)
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Clear extract by centrifugation (30,000 x g, 4°C, 30 min), aliquot and freeze in liquid N2, store at -80°C.
Center for Structures of Membrane Proteins © 2006
T7-RNA polymerase (T7RNAP) preparation (2 days)
Cell growth, harvest (day 1):
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Inoculate 4 liters of LB in Erlenmeyer culture flasks at a ratio of 1:100 with fresh over night culture of BL21
Star x pAR1219. Incubate at 37°C with vigorous shaking until OD600 of 0.6-0.8. Induce T7RNAP with 1 mM
IPTG and incubate at 37°C with vigorous shaking for 5 h.
Harvest cells by centrifugation (4,500 x g, 4°C, 15 min) and resuspend cell pellet in 120 ml T7 buffer
(30 mM Tris, pH 8, 10 mM EDTA, 50 mM NaCl, 5% Glycerol, 10 mM BME).
Cell lysis:
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Disrupt cells with French press (20,000 psi) or cell disrupter. Adjust the supernatant to final concentration of
2% streptomycin sulfate by gentle stirring and drop wise addition of 10% stock.
Remove precipitated DNA from supernatant by centrifugation (30,000 x g, 4°C, 30 min).
Purification:
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Purify T7RNAP by anion exchange chromatography. Equilibrate Q-Sepharose column (1.6 x 10 cm) with T7
buffer, load supernatant with 1ml/min flow rate.
Elute T7RNAP in gradient from 50 mM to 500 mM NaCl in 15 column volumes at a 3 ml/min flow rate. T7RNAP
starts to elute at ~150 mM NaCl.
Dialysis:
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T7RNAP fractions are pooled and dialyzed against 2x 100 volumes of dialysis buffer
(10 mM Tris, pH 8, 1 mM EDTA, 10 mM NaCl, 10% glycerol, 1 mM DTT) over night.
Activity test (day 2):
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T7RNAP activity is tested by in vitro transcription and units are determined by comparison with a
commercial control.
Store T7RNAP:
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Store T7RNAP in aliquots in dialysis buffer adjusted to 50% Glycerol at -20°C.
Center for Structures of Membrane Proteins © 2006
List of reaction components
No
Component
Abbreviation
Supplier
Order no
1
Acetyl Phosphate (litium potassium salt)
AcP
Fluka
1409
2
Adenosine-5’triphosphate (disodium salt)
ATP
Roche
127523
3
Bond-Breaker TCEP Solution, neutral pH
TCEP
Pierce
77720
4
Complete (EDTA free)
Complete
Roche
11873580001
5
Cytidine-5’triphosphate (disodium salt)
CTP
Fluka
30320
6
Folinic acid (calcium salt)
FA
Sigma
F-7878
7
Guanosine-5’triphosphate (disodium salt)
GTP
Fluka
51120
8
Posphoenolpyruvic acid (monopotassium salt)
PEP
Applichem
A2271,0005
9
Polyethylenglycol 8000
PEG8000
Sigma
P-4463
10
Pyruvate Kinase (from rabbit muscle)
PK
Roche
109045
11
SUPERase Inhibitor
RNAsin
Ambion
AM2696
12
tRNA E. coli
tRNA
Roche
109550
13
Uridine-5’triphosphate (trisodium salt)
UTP
Fluka
94370
14
20 l-amino acids
AA
Fluka
LAA21
Center for Structures of Membrane Proteins © 2006
Stock solutions of CF components 1
No
Compound
Stock
1
tRNA
40 mg/ml
2
AcP
1M
3
PEP
1M
4
PK
10 mg/ml
5
ATP
6
MW
Volume
In weight
1 ml
40 mg
184.1
1 ml
184 mg
-> pH 7 (+10 µl 10M KOH)
206.1
1 ml
206 mg
-> pH 7 (+235 µl 10M KOH)
360 mM
605.2
1 ml
218 mg
-> pH 7 (+117 µl 5M NaOH)
GTP
240 mM
567.1
1 ml
136 mg
-> pH 7 (+36 µl 5M NaOH)
7
CTP
240 mM
527.1
1 ml
127 mg
-> pH 7 (+72 µl 5M NaOH)
8
UTP
240 mM
550.1
1 ml
132 mg
-> pH 7 (+33 µl 5M NaOH)
9
NTP-Mix
75 x
10
FA
10 mg/ml
511.5
1 ml
10 mg
11
HEPES-KOH
2.5 M
238.3
10 ml
5,958 mg
-> pH 8 (+1350 µl 10M KOH)
12
Mg(oAc)2
2M
214.4
10 ml
4,288 mg
Filter 0.2 µm
13
KoAc
4M
98.13
10 ml
3,925 mg
Filter 0.2 µm
14
PEG 8000
40 %
10 ml
4,000 mg
15
NaN3
10 %
1 ml
100 mg
4 ml
65.91
Center for Structures of Membrane Proteins © 2006
Information
Same volume each NTP
Stock solutions of CF components 2
No
Compound
Stock
MW
Volume
In weight
1 ml
1 Tablet
16
Complete
50 x
17
35
All AAs
except of
Tyrosine
100 mM
36
Tyrosine
20 mM
37
AA mix
4 mM
50 ml
2 ml each AA stock, 10 ml
Tyrosine stock, 2 ml water
38
RCWMDE
16.7 mM
24 ml
4 ml of AAs R, C, W, M, D, E
Solubilized in water,
Tryptophan in 100 mM
HEPES pH 8
15 ml
181.2
50 ml
Information
181.2 mg
All stock solutions are stored at -20°C and thawed before use
Center for Structures of Membrane Proteins © 2006
Pipetting protocol
Final FM (17 ml) -> pre-incubate at 30°C
For FM (17 ml) and RM (1 ml)
No
Component
Stock
Final
added
14
FM part
15
S30 buffer
100 %
40 %
6920 µl
16
AA mix
4 mM
1
2163 µl
17
H2 O
6909 µl
No
Component
Stock
Final
added
1
NaN3
10 %
0.05 %
92 µl
2
PEG 8000
40 %
2%
918 µl
3
KoAc
4M
270 mM
600 µl
4
Mg(oAc)2
2M
14.5 mM
82 µl
5
HEPES-KOH
2.5 M
100 mM
646 µl
No
Component
6
Complete
50 x
1x
367 µl
14
RM part
7
FA
10
mg/ml
0.1
mg/ml
184 µl
18
PK
10mg/ml
40µg/ml
4.2 µl
8
TCEP
500 mM
2 mM
73 µl
19
tRNA
40
mg/ml
0.5
mg/ml
13.1 µl
9
NTP
75 x
1x
245 µl
20
T7RNAP
400 U/µl
0.5 U/µl
1.3 µl
10
PEP
1M
20 mM
367 µl
21
RNasin
20 U/µl
0.3 U/µl
15.8 µl
11
AcP
1M
20 mM
367 µl
22
S30 extract
100 %
40 %
420.0 µl
12
AA mix
4 mM
0.5 mM
2294 µl
23
Plasmid
1 mg/ml
15µg/ml
15.8 µl
13
RCWMDE
16.7 mM
1 mM
1101 µl
24
H2 O
Center for Structures of Membrane Proteins © 2006
1308 µl
Final RM (1 ml) -> keep on ice
Stock
Final
added
424.7 µl
155.0 µl
Reaction compartments for dialysis mode
Analytical scale (50-100 ul):
Preparative scale (1-3 ml):
Slide-ALyzer MINI
incubate
at 30°C,
200 rpm
100 ul RM
1400 ul FM
75 ul RM
1050 ul FM
Slide-A-Lyzer
3 ml RM
51 ml FM
incubate at
30°C, 200 rpm
50 ul RM
700 ul FM
No
Volume
MWCO
Name
Supplier
Order no
1
50-100 µl
20 kDa
Slide-A-Lyzer MINI
Pierce
69590
2
0.5 – 3 ml
20 kDa
Slide-A-Lyzer
Pierce
66003
Center for Structures of Membrane Proteins © 2006
Cellular vs. CF IMP expression systems
Cloning/DNA preparation / linear PCR fragments
Transformation
Reaction/Fermentation set-up
Reaction set-up
Incubation (overnight – days)
Incubation (15 hrs.)
Induction of expression
Harvest
Cell-disruption
Isolation of membrane fraction
Solubilization of IMPs out of membrane
cellular
Center for Structures of Membrane Proteins © 2006
Purification
(Solubilization of pellet)
cell-free
CF expression of IMPs
(P-CF)
(D-CF)
(L-CF)
P-CF:
insoluble expression,
detergent solubilization
D-CF:
direct translation into
detergent micelles
L-CF:
direct translation into
bicells, lipids, NLPs,
preformed liposomes
3 possible modes for CF IMP expression
Center for Structures of Membrane Proteins © 2006
Klammt et. al 2007: Cell-free expression of
integral membrane proteins for structural
studies. In: Cell-free expression techniques,
A. Spirin and J. Swartz (eds.), Wiley-VCH,
Weinheim, Chapter 8, pp.141-164.
List of detergents/ lipids used with CF system
Detergent
/ Lipid
Name
Working
conc.
x CMC
Mode
Supplier
1
LMPG
1-Mysteroyl-2-hydroxy-sn-Glycero-3-(phospho-rac-(1-glycerol))
1 %
420
P-CF
Avanti
2
LPPG
1-Palmitoyl-2-hydroxy-sn-Glycero-3-(phospho-rac-(1-glycerol))
1%
n.a.
P-CF
Avanti
3
FC12
Dodecylphosphocholine
1%
19
P-CF
Anatrace
4
SDS
Sodiumdodecylsulfate
1%
4.2
P-CF
Sigma
5
Brij-35
Polyoxyethylene-(23)-lauryl-ether
0.1 %
10.4
D-CF
Sigma
6
Brij-58
Polyoxyethylene-(20)-cetyl-ether
1.5 %
178
D-CF
Sigma
7
Brij-78
Polyoxyethylene-(20)-stearyl-ether
1%
189
D-CF
Sigma
8
Brij-98
Poyoxyethylene-(20)-oleyl-ether
0.2 %
70
D-CF
Sigma
9
Digitonin
Digitonin
0.4 %
4.5
D-CF
Sigma
10
DDM
n-Dodecyl-b-D-maltoside
0.1 %
15
D-CF
Anatrace
11
TX-100
PEG P-1,1,3,3-tetra-methyl-butylphenyl-ether
0.2 %
13.4
D-CF
Sigma
12
ECL
E. coli lipid mixture
0.4 %
-
L-CF
Avanti
13
DMPC
1,2-Dimyristoyl-sn-glycero-3-phosphocholine
0.4 %
-
L-CF
Avanti
14
DOPC
1,2-Dioleyl-sn-glycero-3-phosphocholine
0.4 %
-
L-CF
Avanti
15
DSPC
1,2-Distaeroyl-sn-glycero-3-phosphocholine
0.4 %
-
L-CF
Avanti
No
Center for Structures of Membrane Proteins © 2006
Trouble shooting
No IMP product formation
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Verify efficiency of CF system by expression controls (e.g., with GFP)
Ensure high quality of template DNA
Modify DNA template design (e.g., addition of expression tags)
Analyze mRNA secondary structures to ensure efficient initiation and elongation
of translation
Ensure compatibility of extra compounds supplied with CF system
Low IMP expression levels
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Optimize Mg2+ and K+ ion concentration
Adjust amino acid composition
Increase tRNA concentration
Try potentially beneficial additives
Low IMP solubilization in D-CF
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Evaluate a series of detergent types
Optimize the final detergent concentration
Provide detergent mixtures or combinations of detergents/lipids
Decrease expression temperature
Production of IMP fragments
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Stabilize IMP by protease inhibitors during CF expression
Increase tRNA concentration
Use S30 extracts from protease-negative strains
Center for Structures of Membrane Proteins © 2006
N-CF – a new mode for CF expression of IMPs
The polymer NVoy (Expedeon) enables soluble CF expression
and analysis of functional GPCRs
Analyzed GPCRs in the presence of NVoy are homogenous,
functional and can be analyzed by NMR
ligand
binding
N-CF expression of
diverse GPCRs
no
aggregation
homogenous
by
SEC-UV/RI/LS
Center for Structures of Membrane Proteins © 2006
preliminary
NMR data
Recommended references
Klammt et al. 2004 (EJB)
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P-CF protocol for CF IMP expression
Klammt, Schwarz et al. 2005 (FEBS J)
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detailed D-CF protocol for different IMPs
Klammt et al. 2006 (FEBS J)
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detailed review about CF IMP in P-CF, D-CF
Klammt et al. 2007 (MMB)
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detailed expression protocols for extract preparation and P-CF and D-CF set-up
Schwarz et al. 2007 (Nature Prot)
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detailed expression protocols for extract and T7RNAP preparation and P-CF, D-CF
Klammt et al. 2007 (JSB)
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CF expression of GPCRs
Schwarz et al. 2008 (Proteomics)
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recent review about CF IMP in P-CF, D-CF, L-CF
Klammt et al. 2009 (submitted)
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N-CF expression of functional GPCRs
Center for Structures of Membrane Proteins © 2006