Center for Structures of Membrane Proteins © 2006 Cell-free expression of integral membrane proteins Christian Klammt, Innokentiy Maslennikov, Witek Kwiatkowski

Center for Structures of Membrane Proteins © 2006

Cell-free expression of integral membrane Cell-free expression of integral membrane proteinsproteinsCell-free expression of integral membrane Cell-free expression of integral membrane proteinsproteins

Christian Klammt, Innokentiy Christian Klammt, Innokentiy Maslennikov, Maslennikov, Witek Kwiatkowski and Senyon ChoeWitek Kwiatkowski and Senyon ChoeThe Salk Institute, Structural Biology Laboratory, La Jolla, The Salk Institute, Structural Biology Laboratory, La Jolla, CACA


OutlineOutlineCell-free (CF) expression systems– Overview (extract sources, reaction set-ups, commercial

systems)

Protocols for set-up of an E. coli based CF expression system– 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)– Possible modes for CF IMP expression

• P-CF (insoluble precipitate)• D-CF (detergent)• L-CF (lipids)

– List of detergents/ lipids used with CF system– Trouble shooting

N-CF – a new mode for CF expression of IMPs


CF expression as alternative for difficult proteinsCF expression as alternative for difficult proteins

Membrane proteins

Labeled proteins

Modified proteins

Unstable proteins

Cytotoxic proteins

Metallo proteins

Disulfide bonded proteins

Apo-proteins

DisulfidDisulfide e

bonded bonded proteinsproteins

CFCF

ModifiModified ed

proteiproteinsns

UnstablUnstable e

proteinsproteinsCytotoxiCytotoxic c

proteinsproteins

Metallo Metallo proteinsproteins

Specifically Specifically labeled labeled proteinsproteins

MembranMembrane e

proteinsproteins

Apo-Apo-proteinsproteins

+ m

od

ified

aa

+

isot

opical

ly

labe

led

aa

+ chaperones,

protease

inhib.

+ oxidative cond., PDIs

+ m

etal

ion

of cho

ice

+ cofactors

+ detergents,

lipids


CF extract sources and systemsCF extract sources and systems

Wheat germ– low nuclease activities, long life-times (days), preparative

system– difficult extract preparation, complex system

E. coli – high efficiency, easy extract preparation, optimized protocols

for various applications available, preparative system– high variations, shorter life-times (hours)

Rabbit reticulocyte – eukaryotic system – difficult extract preparation, complex non preparative system

Insect cells– eukaryotic system, post-translational modifications– complex non preparative system


Reaction set-up - batch vs. dialysisReaction set-up - batch vs. dialysis

Easy set-up

Short reaction times (<3 hours)

Agglomeration of harmful LMW products, fast energy consumption

High throughput screening

Up to 1 mg/ml

Need for suitable dialysis set-up

Long reaction times (> 12 hours)

Continuous exchange of harmful LMW products and supply of energy

Up to 6 mg/ml of protein in RM

Limited high throughput screening

Ribosomes, ARSases,

tRNA, Polymerase

s, DNA, mRNA, IFs, EFs, RFs, NTPs,

amino acids, Energy system

SUBSTRATES: amino acids,

NTPs, energy substratesLMW

PRODUCTS: NMPs, PPi, Pi

COUPLED TRANSCRIPTION/

TRANSLATION

batchbatch dialysisdialysis

Dialysis membrane

Reaction mixture (RM)

Feeding mixture (FM)

stirrer


Commercially available systemsCommercially available systems

E. coli: – ExpresswayTM (Invitrogen)– RTS E. coli (Roche)– EasyExpress E.coli (Qiagen)– S30 T7 High-Yield (Promega)

Wheat germ– ENDEXT® (Cell-Free Sciences)– RTS wheat germ (Roche)– TNT® wheat germ (Promega)

Rabbit reticulocyte– TNT® rabbit reticulocyte

(Promega)

Insect– EasyExpress insect (Qiagen)– TNT® insect (Promega)

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 systemE. 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,…

Coupled transcription/translation


E. coli S30 extract preparation (3 days)E. coli S30 extract preparation (3 days)Cell growth (day 1):

– 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 (OD 600 = 5-6). All following steps on ice!

Cell wash:– 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):– 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:– 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:– Supplement extract with 400 mM NaCl (5 M stock) and incubate for 45 min at 42°C.

Dialysis:– Dialyse against 2x 40 volumes over night with first buffer exchange after 2 h into S30 buffer (10 mM

Tris-Acetat, 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)– Clear extract by centrifugation (30,000 x g, 4°C, 30 min), aliquot and freeze in liquid N 2, store at -80°C.


T7-RNA polymerase (T7RNAP) preparation (2 days)T7-RNA polymerase (T7RNAP) preparation (2 days)Cell growth, harvest (day 1):

– 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:– 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:– 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:– 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):– T7RNAP activity is tested by in vitro transcription and units are determined by comparison

with a commercial control.

Store T7RNAP:– Store T7RNAP in aliquots in dialysis buffer adjusted to 50% Glycerol at -20°C.


List of reaction componentsList of reaction components

NoNo ComponentComponent AbbreviatiAbbreviationon

SupplierSupplier Order noOrder no

11 Acetyl Phosphate (litium potassium salt)

AcP Fluka 1409

22 Adenosine-5’triphosphate (disodium salt)

ATP Roche 127523

33 Bond-Breaker TCEP Solution, neutral pH

TCEP Pierce 77720

44 Complete (EDTA free) Complete Roche 11873580001

55 Cytidine-5’triphosphate (disodium salt)

CTP Fluka 30320

66 Folinic acid (calcium salt) FA Sigma F-7878

77 Guanosine-5’triphosphate (disodium salt)

GTP Fluka 51120

88 Posphoenolpyruvic acid (monopotassium salt)

PEP Applichem

A2271,0005

99 Polyethylenglycol 8000 PEG8000 Sigma P-4463

1010 Pyruvate Kinase (from rabbit muscle) PK Roche 109045

1111 SUPERase Inhibitor RNAsin Ambion AM2696

1212 tRNA E. coli tRNA Roche 109550

1313 Uridine-5’triphosphate (trisodium salt)

UTP Fluka 94370

1414 20 l-amino acids AA Fluka LAA21


Stock solutions of CF components 1Stock solutions of CF components 1

NNoo

CompounCompoundd

StockStock MWMW VolumVolumee

In weightIn weight InformationInformation

11 tRNA 40 mg/ml 1 ml 40 mg

22 AcP 1 M 184.1 1 ml 184 mg -> pH 7 (+10 µl 10M KOH)

33 PEP 1 M 206.1 1 ml 206 mg -> pH 7 (+235 µl 10M KOH)

44 PK 10 mg/ml

55 ATP 360 mM 605.2 1 ml 218 mg -> pH 7 (+117 µl 5M NaOH)

66 GTP 240 mM 567.1 1 ml 136 mg -> pH 7 (+36 µl 5M NaOH)

77 CTP 240 mM 527.1 1 ml 127 mg -> pH 7 (+72 µl 5M NaOH)

88 UTP 240 mM 550.1 1 ml 132 mg -> pH 7 (+33 µl 5M NaOH)

99 NTP-Mix 75 x 4 ml Same volume each NTP

1010 FA 10 mg/ml 511.5 1 ml 10 mg

1111 HEPES-KOH

2.5 M 238.3 10 ml 5,958 mg -> pH 8 (+1350 µl 10M KOH)

1212 Mg(oAc)2 2 M 214.4 10 ml 4,288 mg Filter 0.2 µm

1313 KoAc 4 M 98.13 10 ml 3,925 mg Filter 0.2 µm

1414 PEG 8000 40 % 10 ml 4,000 mg

1515 NaN3 10 % 65.91 1 ml 100 mg


Stock solutions of CF components 2Stock solutions of CF components 2

NNoo

CompounCompoundd

StockStock MWMW VolumVolumee

In weightIn weight InformationInformation

1616 Complete 50 x 1 ml 1 Tablet

1717- -

3535

All AAs except of Tyrosine

100 mM

15 ml

Solubilized in water, Tryptophan in 100 mM HEPES pH 8

3636 Tyrosine 20 mM 181.2 50 ml 181.2 mg

3737 AA mix 4 mM 50 ml 2 ml each AA stock, 10 ml Tyrosine stock, 2 ml water

3838 RCWMDE 16.7 mM 24 ml 4 ml of AAs R, C, W, M, D, EAll stock solutions are stored at -20°C and thawed

before use


Pipetting protocolPipetting protocol

NoNo ComponeComponentnt

StockStock FinalFinal addedadded

11 NaN3 10 % 0.05 % 92 µl

22 PEG 8000 40 % 2 % 918 µl

33 KoAc 4 M 270 mM

600 µl

44 Mg(oAc)2 2 M 14.5 mM

82 µl

55 HEPES-KOH

2.5 M 100 mM

646 µl

66 Complete 50 x 1 x 367 µl

77 FA 10 mg/ml

0.1 mg/ml

184 µl

88 TCEP 500 mM

2 mM 73 µl

99 NTP 75 x 1 x 245 µl

1010 PEP 1 M 20 mM 367 µl

1111 AcP 1 M 20 mM 367 µl

1212 AA mix 4 mM 0.5 mM 2294 µl

1313 RCWMDE 16.7 mM

1 mM 1101 µl



1414 FM part 6909 µl

1515 S30 buffer

100 % 40 % 6920 µl

1616 AA mix 4 mM 1 2163 µl

1717 H2O 1308 µl



1414 RM part 424.7 µl

1818 PK 10mg/ml

40µg/ml

4.2 µl

1919 tRNA 40 mg/ml

0.5 mg/ml

13.1 µl

2020 T7RNAP 400 U/µl

0.5 U/µl

1.3 µl

2121 RNasin 20 U/µl 0.3 U/µl

15.8 µl

2222 S30 extract

100 % 40 % 420.0 µl

2323 Plasmid 1 mg/ml

15µg/ml

15.8 µl

2424 H2O 155.0 µl

For FM (17 ml) and RM (1 ml)For FM (17 ml) and RM (1 ml)

Final FM (17 ml) Final FM (17 ml) -> pre-incubate at 30°C-> pre-incubate at 30°C

Final RM (1 ml) Final RM (1 ml) -> keep on ice-> keep on ice


Reaction compartments for dialysis modeReaction compartments for dialysis mode

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

Analytical scale (50-100 ul): Preparative scale (1-3 ml):

100 ul RM1400 ul FM

75 ul RM1050 ul FM

50 ul RM700 ul FM

Slide-A-Lyzer3 ml RM51 ml FM

incubate at 30°C, 200 rpm

Slide-A-Lyzer MINIincubate at 30°C, 200 rpm


Cellular vs. CF IMP expression systemsCellular vs. CF IMP expression systems

Cloning/DNA preparation / linear PCR fragments

Transformation

Incubation (overnight – days)

Induction of expression

Harvest

Cell-disruption

Isolation of membrane fraction

Solubilization of IMPs out of membrane

Purification

Reaction set-up

Incubation (15 hrs.)

(Solubilization of pellet)

Reaction/Fermentation set-up

cellularcellular cell-freecell-free


CF expression of IMPsCF expression of IMPs

P-CF: insoluble expression, detergent solubilization

D-CF: direct translation into detergent micelles

L-CF: direct translation into bicells, lipids, NLPs, preformed liposomes

(P-CF) (D-CF) (L-CF)

3 possible modes for CF IMP expression

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 systemList of detergents/ lipids used with CF system

NNoo

DetergeDetergent/ Lipidnt/ Lipid

NameName

WorkiWorking ng conc.conc.

x x CMCCMC

ModMode e

SupplieSupplierr

11 LMPG 1-Mysteroyl-2-hydroxy-sn-Glycero-3-(phospho-rac-(1-glycerol))

1 % 420 P-CF Avanti

22 LPPG 1-Palmitoyl-2-hydroxy-sn-Glycero-3-(phospho-rac-(1-glycerol))

1 % n.a. P-CF Avanti

33 FC12 Dodecylphosphocholine 1 % 19 P-CF Anatrace

44 SDS Sodiumdodecylsulfate 1 % 4.2 P-CF Sigma

55 Brij-35 Polyoxyethylene-(23)-lauryl-ether 0.1 % 10.4 D-CF

Sigma

66 Brij-58 Polyoxyethylene-(20)-cetyl-ether 1.5 % 178 D-CF

Sigma

77 Brij-78 Polyoxyethylene-(20)-stearyl-ether 1 % 189 D-CF

Sigma

88 Brij-98 Poyoxyethylene-(20)-oleyl-ether 0.2 % 70 D-CF

Sigma

99 Digitonin

Digitonin 0.4 % 4.5 D-CF

Sigma

1010 DDM n-Dodecyl--D-maltoside 0.1 % 15 D-CF

Anatrace

1111 TX-100 PEG P-1,1,3,3-tetra-methyl-butylphenyl-ether 0.2 % 13.4 D-CF

Sigma

1212 ECL E. coli lipid mixture 0.4 % - L-CF Avanti

1313 DMPC 1,2-Dimyristoyl-sn-glycero-3-phosphocholine 0.4 % - L-CF Avanti

1414 DOPC 1,2-Dioleyl-sn-glycero-3-phosphocholine 0.4 % - L-CF Avanti

1515 DSPC 1,2-Distaeroyl-sn-glycero-3-phosphocholine 0.4 % - L-CF Avanti


Trouble shootingTrouble shootingNo IMP product formation

– 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– Optimize Mg2+ and K+ ion concentration– Adjust amino acid composition– Increase tRNA concentration– Try potentially beneficial additives

Low IMP solubilization in D-CF– 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– Stabilize IMP by protease inhibitors during CF expression– Increase tRNA concentration– Use S30 extracts from protease-negative strains


N-CF – a new mode for CF expression of IMPsN-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

N-CF expression ofdiverse GPCRs

ligand binding

noaggre-gation

homo-genous bySEC-UV/RI/LS

preliminaryNMR data


Recommended referencesRecommended referencesKlammt et al. 2004 (EJB)

– P-CF protocol for CF IMP expression

Klammt, Schwarz et al. 2005 (FEBS J)– detailed D-CF protocol for different IMPs

Klammt et al. 2006 (FEBS J)– detailed review about CF IMP in P-CF, D-CF

Klammt et al. 2007 (MMB)– detailed expression protocols for extract preparation and P-CF and D-

CF set-up

Schwarz et al. 2007 (Nature Prot)– detailed expression protocols for extract and T7RNAP preparation and

P-CF, D-CF

Klammt et al. 2007 (JSB)– CF expression of GPCRs

Schwarz et al. 2008 (Proteomics)– recent review about CF IMP in P-CF, D-CF, L-CF

Klammt et al. 2009 (submitted)– N-CF expression of functional GPCRs

Documents

Center for Structures of Membrane Proteins © 2006 Cell-free expression of integral membrane proteins Christian Klammt, Innokentiy Maslennikov, Witek Kwiatkowski