Frank Bernhard

Institute of Biophysical Chemistry

University of Frankfurt, Germany

Cell-Free Expression of Membrane Proteins in Artificial Environments

4th P4EU Meeting Porto 2013

Coupled Transcription-Translation

- Bacterial S30 extract

Removal of endogenous amino acids

Elimination of endogenous mRNA

- T7-RNA polymerase

- Template DNA (T7-regulatory sequences),

e.g. pET-vectors, pIVEX

- tRNA

- Amino acids

- RNase, protease inhibitors

- Energy system: acetyl/creatine phosphate,

pyruvate, kinases

- NTPs

- Suitable buffer system

- Mg2+-, K+-Ions

Optional additives:

I. Efficiency enhancer: PEG, spermidine, cAMP, oxalate, coenzyme A, NAD…

II. Chemical chaperones: Glycerol, polysaccharides, alcohols…

III. Protein chaperones: GroEL/ES…

IV. Target specific stabilizers: Ligands, cofactors, inhibitors, subunits…

V. Solubilization compounds: Detergents, lipids, surfactants…

VI. Redox systems: DTT, glutathione system, DsbA/C…

Applications of cell-free expression

I. High throughput, protein expression in artificial environments

Proteome expression screens by robotic devices, X-ray, NMR!, reducing inclusion body formation by addition of stabilizers

II. Expression of disulfide-bridged proteins

Venome peptides, antibodies…

Modulation of oxidizing/reducing conditions

III. Co-expression of multisubunit complexes

Identical vectors (no care about compatibility, copy number..)

Pre-expression, co-expression

IV. Excellent system for protein labelling

Fluorescence enhanced amino acids, seleno-methionine, stable isotopes

V. Expression of proteins that cannot be obtained in living hosts Toxins, unstable proteins, apo-enzymes, artificial cofactor incorporation

membrane proteins

Cell-Free Membrane Protein Expression

Continuous exchange cell-free system

(CECF)

Method Development

Reckel et al (2010) Methods Mol. Biol.

Kai et al (2011) Methods Mol. Biol.

Haberstock et al (2012) Prot. Expr. Purif.

Junge et al (2010) New Biotechnol.

Junge et al (2008) Cell. Mol. Life Sci.

Sobhanifar et al (2010) J. BioMol. NMR

Reckel et al (2008) PNAS

Schwarz et al (2008) Proteomics

Roos et al (2012) Mol. Membr. Biol.

Feeding Mix

Reaction Mix

Maxi-CECF Reactor

Reaction

Mix

Feeding Mix

Mini-CECF Reactor

Cloning/DNA preparation / linear PCR fragments

Transformation

Incubation (overnight – days)

Induction of expression

Harvest

Cell-disruption

Isolation of membrane fraction

Extraction of MPs out of membrane

Purification

Reaction set-up

Incubation (overnight)

(Solubilization 1-3 hrs)

Reaction/Fermentation set-up

Cellular - Cell-Free Membrane Protein Expression

Cellular Cell-free

Targeting

Translocation

Toxicity

Proteolysis Vector issues

Cell handling

Media

Sterility

Fusions

Promoter

Regulation/Induction

+ efficient labelling

+ individualized protocols

+ artificial hydrophobic

environments

I: → Yield = Basic expression protocol: P-CF

Tag variation, template design and quality, codon usage

Basic compounds (ions, extract source, energy system)

RM:FM ratio, re-feeding of substrates

Systematic 3-Level Protocol Development

Junge et al., (2011) New Biotechnology 28:262-71.

Schwarz et al., (2008) Proteomics 8:3933-46.

Junge et al., (2008) Cell. Mol. Life Sci. 65:1729-55.

II: → Solubility = Array of expression protocols: D-CF + L-CF

Type and concentration of hydrophobic compound(s), detergents, lipids,

D/L-mixtures, bicelles, liposomes, nanodiscs

III: → Quality = Selection of one/few individual protocols

Stability, homogeneity, activity

Detergent exchange; 1st and 2nd detergent, additives, stabilizer

Quality screening of sample arrays

Customized target specific production protocols

Roos et al. (2012) MolMemBiol.

Monitoring of Cell-Free Membrane Protein Expression

130 Targets

80 % success,

Size: > 100 kDa, 15 TMS

Schwarz et al. (2010) Proteomics 10: 1762-79

E. coli cell-free system: Purification

Single step purification with affinity chromatography

Reaction mix / resolubilized pellet can be loaded directly on the affinity matrix

G-Protein Coupled Receptors (GPCRs)

Junge et al. (2010) J. Struct. Biol. 172: 94-106

Junge et al. (2008) Cell. Mol. Life Sci. 65: 1729-55

Klammt et al. (2007) J. Struct. Biol. 158: 482-93

Klammt et al. (2007) FEBS J. 274: 3257-69

bovine

Rhodopsin

rat

Corticotropin

releasing

factor

human

Vasopressin

type 2

Cell-free Expression Modes for Membrane Protein Synthesis

► 3 basic ways to produce soluble membrane proteins

DL-CF

P-CF Expression Mode

● Spin down

● Wash precipitate

● Option: Selective solubilization

● Option: Complete Solubilization (1 h, RT)

● Affinity purification, e.g. Ni-NTA

Km(MPP+) = 35 µM (30 µM*)

turnover number = 19 sec-1 (20 sec-1*) MPP:1-methyl-4-phenyl-pyridinium

*rOCT1 from insect cells

180

130

100

73

54

48

35

24

1 2 3

rOCT1

180

130

100

73

54

48

35

24

1 2 3

rOAT1

Eukaryotic Organic Cation/Anion Transporters

Cooperation: T. Keller/H. Koepsell

Cation selectivity of rOCT1 IC50:

TEA+: 159 µM / 196 µM*

TPeA+: 2.9 µM / 1.8 µM*

TBuA+: 20 µM / 19 µM*

Keller et al. (2008) Biochemistry 47: 4552-4564

Keller et al., (2011) J. Biol. Chem. 286: 37874-86

P-CF

Presenilin 1(PS1): 53 kDa

Presenilin-NTF (NTF): 31 kDa

Presenilin-CTF (CTF): 20 kDa

Nicastrin: 78 kDa

Pen-2: 12 kDa

Aph-1: 27 kDa

Sobhanifar et al. (2010), PNAS 107, 9644-9649

Sobhanifar et al. (2010), J Biomol NMR 46, 33-43

Solution NMR structure of P-CF expressed presenilin-1 CTF

Positron Research Group B. Pucci / A. Polidori J. L. Popot

L-CF D-CF

Cell-free expression of GnRHR

Test different hydrophobic environments

Nanodiscs

DMPC

DMPG

DOPC

DPPC

DOPC+DPPC

POPC

Detergents Amphipol: A8-35

D-CF:

Brij-35

Brij-58

Brij-78

Solubilization of P-CF

precipitates:

LMPG

LMPC

DDM

DPC

Ligand binding studies: MST, Biacore, Pull-down

Soluble expression in D-CF mode

P-CF Brij35 Brij58 Brij78 DDM DPC Digitonin DHPC

M RM

PR from Digitonin in DHPC, Superdex200

-10

0

10

20

30

40

50

60

70

0 0.5 1 1.5 2 2.5 3

elution volume [ml]

ab

so

rpti

on

[m

AU

]

-10

-5

0

5

10

15

20

25

30

35

40ab

so

rpti

on

[m

AU

]280 nm

380 nm

535 nm

Sina Reckel

Throughput Cell-Free Yield + Solubility + Quality

Optimization Screening

Brij58 DPC P-CF DMPC Brij58 + DMPC Triton + DMPC

Digitonin : diC7PC 4:1

Reckel et al. (2011) Angew. Chem. Intl. Ed.

Quality Screening of Endothelin-A Receptor Samples

P-CF

P-CF

D-CF

Junge et al. (2010) J. Struct. Biol. 172: 94-106

T. Meier

D. Matthies

MPI of Biophysics

Frankfurt/Main

Cell-Free Expression of Multisubunit Complexes

F1F0 ATP Synthase

3 membrane protein subunits

5 soluble protein subunits

25 protomers, 542 kDa

Cell-Free Expression of Multisubunit Complexes

Matthies et al. (2011) J. Mol. Biol. 413: 593-603

In vivo expression Cell-free expression

Adapted from J. Popot et al. 2011

Nonionic APol

A8-35

Amphipols

Roos et al (2012) Mol Membr Biol

Proverbio et al (2013) BBA

L-CF Expression Mode

Adapted from Nath et al. 2006

Nanodiscs

Nanodiscs Liposomes Consistence between + - preparations Monodispersity + - Time and temperature + + stability of MP Access form both sides + - of the membrane Quantification of + (+)‏ protein insertion Purification‏of‏“loaded” + (+)‏ bilayers only

Insertion of CF produced MPs into

Nanodiscs

Lipid MSP1 :: Lipid MSP1E3 :: Lipid

DMPC 1:80 1:115

POPC 1:55 1:85

DOPC 1:30 1:80

DMPG 1:70 1:110

DOPG 1:30 1:90

DOPE 1:30 1:80

PC Soybean 1:40 1:60

PL E. coli 1:40 1:60

TL E. coli 1:40 1:50

PL Brain 1:45 1:70

TL Brain 1:35 1:60

Co-translational insertion into preformed nanodiscs

No detergent contact of membrane protein Roos et al (2012) BBA

In Vitro Nanodisc Formation

DOPC nanodisc

0

50

100

150

200

250

0,0

0

0,1

10

,23

0,3

40

,46

0,5

70

,68

0,8

00

,91

1,0

2

1,1

41

,25

1,3

71

,48

1,5

91

,71

1,8

21

,93

2,0

52

,16

2,2

82

,39

2,5

02

,62

2,7

3

2,8

52

,96

3,0

73

,19

3,3

03

,41

3,5

33

,64

DOPC 1:60

DOPC 1:80

DOPC 1:100

Best: 1:60

0

50

100

150

200

250

0,0

0,1

0,3

0,4

0,5

0,7

0,8

0,9

1,0

1,2

1,3

1,4

1,6

1,7

1,8

2,0

2,1

2,2

2,4

2,5

2,6

2,7

2,9

3,0

3,1

3,3

3,4

3,5

3,7

Volume (ml)

DPPC NDs

DPPC 1:100

DPPC 1:150

DPPC 1:170

DPPC 1:200

Best: 1:100

0

50

100

150

200

250

0,0

0,1

0,2

0,3

0,5

0,6

0,7

0,8

0,9

1,0

1,1

1,3

1,4

1,5

1,6

1,7

1,8

1,9

2,0

2,2

2,3

2,4

2,5

2,6

2,7

2,8

3,0

3,1

3,2

3,3

3,4

3,5

3,6

DOPC:DPPC 1:30:60

DOPC:DPPC 1:40:80

DOPC:DPPC 1:40:40

DOPC-DPPC NDs

Best: 1:40:40 (or 1:30:60)

SugE

Roos et al (2012) BBA

Proteorhodopsin

MraY Translocase

Optimization of Nanodisc Complex Formation

Zocher et al. (2011) ACS Nano

Single Molecule Force Microscopy of Bacteriorhodopsin

in Nanodiscs

Bacteriorhodopsin inserts as a

Trimer

Roos et al (2012) BBA

Laser flash induced time-dependent absorbance changes of green

proteorhodopsin reconstituted from DHPC into DMPC/DMPA

proteoliposomes and DMPC/DMPA nanodiscs. The PR-nanodisc sample

shows a spectral behavior similar to that of proteoliposomes.

Moers et al. (2013) BBA

Ranaghan et al., JACS 2011

Functional Analysis of PR/Nanodisc Complexes

Ligand nM IC50 (IC50 literature)

ET-1 8 (0.3)

Ala-4-ET-1 2663 (> 2000)

BQ-123 38 (1.4)

IRL-1620 300 (> 500)

Ligand nM IC50 (IC50 literature)

ET-1 9 (0.2)

Ala-4-ET-1 4 (0.33)

BQ-123 1395 (> 1500)

IRL-1620 0.005 (0.1-56)

ETA: ET-1 = BQ-123 >> ET-3, 4-Ala-ET-1, IRL-1620

ETB: ET-1 = ET-3, 4-Ala-ET-1, IRL-1620 >> BQ-123

Kd ET-1: 1.7 (0.4-0.8)

Kd ET-3: 3400 (>2000)

Kd ET-1: 0.4-0.7 (0.1-0.8)

Kd ET-3: 0.2 (0.1-0-8)

Differential Ligand Binding of GPCR/Nanodisc Complexes

Proverbio et al. (2013) BBA

{

Biosynthesis pathway of Lipid II in E.coli

Cell Wall Biosynthesis in Bacteria

Biosynthesis of Peptidoglycan Precursor

Published in: Tetsuya Tanino; Bayan Al-Dabbagh; Dominique Mengin-Lecreulx; Ahmed Bouhss; Hiroshi Oyama; Satoshi Ichikawa; Akira Matsuda;

J. Med. Chem. 2011, 54, 8421-8439.

DOI: 10.1021/jm200906r

Copyright © 2011 American Chemical Society

Lipid Requirements of E. coli MraY Translocase

Roos et al (2012) BBA

Ma et al (2011) JBC

Goethe University, Biophysical Chemistry • Volker Dötsch

• Natascha Rogova (Expression screening, extract preparation)

• Davide Proverbio, Erika Orbán, Ralf Rues, Fang Dong (GPCRs)

• Erik Henrich (L-CF, nanodiscs, translocase)

• Christopher Hein (Site specific labelling)

• Former Members: Edith Buchinger, Stefan Haberstock, Frederike Junge,

Lei Kai, Yi Ma, Sina Reckel, Christian Roos, Solmaz Sobhanifar

Thomas Meier Klaus Fendler

Vladimir Shirokov Alexander Spirin

Clemens Glaubitz Rupert Abele Joseph Wachtveitl

Hermann Koepsell

Hans-Georg Sahl Tanja Schneider

Stefan Kubick

Martin Caffrey Coilin Boland

Daniel Müller

Alain Milon

Bernard Mouillac

Core Center for Cell-Free Expression

www.structuralbiology.eu