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Michael Lafontaine and C. Roy D. Lancaster



Table 1 Buffers for the purification of the QFR of W. succinogenes

Lysis buffer AEX W1

AEX E1

AEX W2

AEX E2



SEC



50 mM Tris 50 mM Tris 50 mM Tris 50 mM Tris 50 mM Tris 20 mM

pH 7.35

pH 7.35

pH 7.35

pH 7.35

pH 7.35

HEPES pH

7.3

2 mM

Malonate



2 mM

Malonate



2 mM

Malonate



2 mM

Malonate



2 mM

Malonate



20 mM

Malonate



1 mM

DTT



1 mM

DTT



1 mM

DTT



1 mM DTT 1 mM DTT –











1 M NaCl











0.05%

Triton

X-100



0.05%

Triton

X-100



0.1% DM

0.1% DM

0.1% DM

+ 0.01% LM + 0.01% LM + 0.01% LM



0.3 M NaCl 1 mM

EDTA



Composition of buffers used for the purification of W. succinogenes’ QFR

is listed in Table 1. Filter-sterilize and degas all buffers. Add detergent and

reducing agents directly before use.



3.3 Cell lysis procedure for the extraction of a membrane

protein

Resuspend cells in lysis buffer to a 10–30% (w/v) solution and homogenize. Add 1 mM PMSF, 1 mM DTT, and a few microliter of DNAse

(10 mg/mL). Cell lysis is performed with an EmulsiFlex-C3 emulsifier

(Avestin, Ottawa, ON, Canada) by three passes at 1400 bar. The membrane fraction is separated from the cytosolic fraction by ultracentrifugation (100,000 Â g, 1 h, 4 °C). Discard the supernatant and resuspend the

membrane pellet in approximately 100–120 mL 1 Â Tris/malonate

buffer supplied with 1 mM DTT and add Triton X-100 (5% of cell

weight). Stir under nitrogen atmosphere for 30 min at room temperature

and remove unsolubilized material by ultracentrifugation (100,000 Â g,

45 min).



3.4 Cell lysis procedure for the extraction of a periplasmic

protein

Purification of a periplasmic membrane-associated protein is illustrated in

the example of the methylfumarate reductase SdhABE complex. Although



Wolinella succinogenes Membrane Protein Production



113



the enzyme is membrane associated, most of specific activity is found in the

soluble fractions and not in the membranes. This finding together with prediction studies points toward a periplasmic membrane-associated E-type

SQOR ( Juhnke et al., 2009).

For extraction of the SdhABE complex, thaw and flash-freeze 5–10 g

cells in liquid nitrogen for three times and finally resuspend in 2 mL/g

wet cell weight prechilled, anoxic buffer containing 50 mM Tris, pH

8.25, 1 mM malonate, and 1 mM DTT. After pelleting membranes and cell

debris (36,000 Â g, 15 min, 4 °C), add 0.1% dodecyl-β-D-maltoside (w/v) to

the supernatant and stir in a septum flask for 30 min at room temperature

under continuous purging by argon gas. Pellet unsolubilized material

(>200,000 Â g, 1 h). Purge supernatant again by argon gas and apply to

anion exchange chromatography.



3.5 Anion exchange chromatography

As long as there are no affinity tags fused to the produced protein, ion

exchange chromatography provides a valuable method of purifying the protein of interest to homogeneity. In the case of membrane proteins like the

QFR, this method is also used for detergent exchange. After solubilization,

the protein containing supernatant is loaded onto a 300 mL XK column

packed with DEAE Sepharose Fast Flow equilibrated in anaerobic AEX

ă kta purifier FPLC system. After extensive washing to

W1 buffer on an A

remove unspecifically bound proteins, elution is performed with a linear

gradient or in a stepwise fashion to a salt concentration of 1 M NaCl

(AEX E1 buffer). To improve purity, the salt concentration was reduced

to 0.3 M NaCl. The QFR of W. succinogenes elutes at sodium chloride

concentrations of between 100 and 120 mM. After concentrating the elution fraction to approximately 20 mL in a stirred ultrafiltration cell

(Model 8200, Millipore), the QFR containing elution fractions is diluted

with AEX W1 to a final salt concentration of approximately 80–100 mM

NaCl. Subsequently, the protein is loaded on a second DEAE column with

a column volume of 100 mL equilibrated in AEX W1 buffer. The column

with the bound protein is washed with several column volumes of

buffer containing 0.1% (w/v) β-decyl-maltoside and 0.01% (w/v)

β-dodecyl-maltoside (AEX W2 buffer) until the absorbance reaches baseline.

Elution is performed by applying a linear gradient up to 300 mM NaCl (AEX

E2 buffer).



114



Michael Lafontaine and C. Roy D. Lancaster



3.6 Size-exclusion chromatography

After concentrating the elution fraction of the second anion exchange

chromatography with a centrifugal filter concentrator to a volume of

approximately 5–10 mL, the sample is injected onto a size-exclusion chromatography column with the appropriate molecular weight separation range

from 10 kDa to 600 kDa (e.g., Superdex 200 16/60 or 26/60) equilibrated

with SEC buffer. Monodisperse fractions are pooled, concentrated, and

flash-frozen with liquid nitrogen.



3.7 Determination of protein concentration

For measuring protein concentration, several popular methods can be applied

like absorbance measurement at 280 nm, Bradford assay (Bradford, 1976),

or BCA assay (Smith et al., 1985). The latter is used to determine protein

concentration of the QFR as detergent interferes with both the absorbance

measurement at 280 nm and the Bradford assay. The BCA assay is performed

as described in the manufacturer’s manual (Pierce™ BCA Protein Assay Kit,

Pierce Biotechnology, Rockford, IL, USA).



3.8 Functional characterization

Functional characterization of proteins is performed in many ways. In this

chapter, the functional characterization of the QFR of W. succinogenes is

demonstrated on measuring fumarate reductase or quinol oxidation activity

(Unden & Kr€

oger, 1981). This can be performed either in detergentsolubilized state (Lancaster et al., 2000) or in liposomes (Biel et al., 2002;

Madej et al., 2006). The latter method was used to provide experimental

evidence for the “E-pathway” (Lancaster, 2002b) for essential transmembrane proton transfer in the QFR of W. succinogenes by incorporating a

“E-pathway”-defective variant into proteoliposomes (Madej et al., 2006).



3.9 Reconstitution of enzymes in proteoliposomes

Reconstitution of purified enzymes of the anaerobic respiratory chain in

proteoliposomes is performed according to Biel et al. (2002) and Madej

et al. (2006) and comprises two steps: the preparation of sonicated liposomes

and the proteoliposome reconstitution procedure. This procedure ensures a

unidirectional incorporation of the enzyme into the liposomal membrane

where the hydrophilic A and B subunits point outward.



Wolinella succinogenes Membrane Protein Production



115



3.9.1 Material

– Bio-Beads SM-2 (Bio-Rad)

– Chloroform/methanol solution (2:1, v/v): Mix 6 mL chloroform with

3 mL methanol

– n-Dodecyl-β-D-maltopyranoside (Anatrace)

– HEPES–KCl: HEPES (5 mM) pH 7.5, KCl (100 mM)

– Menaquinol (0.1 M) in ethanol (100%)

– 1,2-Dipalmitoyl-sn-glycero-3 phosphocholine (phosphatidylcholine)

and 2-dipalmitoyl-sn-glycero-3-phosphate (phosphatidate) (Avanti Polar

Lipids, Inc.)

3.9.2 Preparation of liposomes

For 5 mL of a 10Â liposome stock solution:

1. Weight in 50 mg phosphatidylcholine and 5 mg phosphatidate directly

into a 100-mL septum flask.

2. Dissolve lipids in 9 mL of a chloroform/methanol mixture (2:1, v/v)

under stirring at room temperature.

3. Add 18.5 μL of a menaquinol stock solution (100 mM) to incorporate a

quinol into the liposomal membrane.

4. Tightly close the septum flask and evaporate solvents completely by

evacuating.

5. Resuspend the dry phospholipid film (layer) in 5 mL of HEPES/KCl

buffer and pour the liposome suspension (11 g phospholipids/L) into a

15-mL Falcon.

6. Sonicate the liposome suspension on ice until it is completely clear

(Bandelin Sonopuls equipped with microtip, 4 °C, 30 W, 40% cycle).

7. Make 1:10 dilutions with HEPES/KCl buffer in 5 mL volume in a new

Falcon (1.1 g phospholipids/L).

8. Store liposomes at À20 °C until further use.

3.9.3 Preparation of proteoliposomes

9. Thaw 1 Â liposome suspension and sonificate on ice for 10–15 min

(Bandelin Sonopuls equipped with microtip, 4 °C, 30 W, 40% cycle).

10. Transfer liposome suspension into a new 10-mL septum flask and add

0.8 g β-dodecyl-maltoside/g phospholipid and stir carefully for 3 h at

room temperature.

11. Add enzyme (0.18 mg/mg phospholipids) stepwise under constant

stirring for 1 h at room temperature.



116



Michael Lafontaine and C. Roy D. Lancaster



12. Remove detergent by addition of Bio-Beads SM-2 (240 mg/mL)

under stirring for 1 h.

13. Transfer liposome solution into a new Falcon and remove Bio-Beads

by centrifugation (5000 Â g, 30 s) and/or filter-sterilize (0.2 μm

pore size).

14. Concentrate proteoliposomes by centrifugation at 17,000 Â g for

1 min.

15. Discard supernatant and resuspend pellet in 50–100 μL HEPES/

KCl buffer.



3.10 Enzymic assays

3.10.1 Material

– UV–VIS Diode Array Spectrophotometer equipped with a multicell

transport connected to a water bath (e.g., 8453 UV–VIS Diode Array

System Agilent Technologies Inc., Santa Clara, CA, USA)

– 25- or 50-μL and 1-mL Microliter syringes with needle (e.g., Hamilton

Bonaduz AG, Bonaduz, Swiss)

– 1-mL Quartz cuvettes (e.g., macro cells, Agilent Technologies Inc., Santa

Clara, CA, USA)

– Airtight plugs (Rotilabo plugs, Roth, Karlsruhe, Germany)

– Potassium phosphate buffer (50 mM), pH 7.4: Filter-sterilize and degas in

a septum flask

– Tris buffer (50 mM), pH 8.0: Filter-sterilize and degas in a septum flask

– 2,3-Dimethyl-1,4-naphthoquinone (DMN) (20 mM): Synthesized as

described (Lancaster et al., 2005)

– Dissolve in ethanol, protect from light, and keep on ice. Do not degas

– Borohydride (20 mg/mL): Degas flask with potassium borohydride

before dissolving in anaerobic ddH2O. Do not degas afterward

– Benzyl viologen (0.1 M): Dissolve in anaerobic ddH2O

– Sodium dithionite (50 mg/mL): Dissolve in ddH2O

– Fumarate (1 M): Dissolve in H2Odest in a septum flask and degas

– Fumarate (0.1 M): Dissolve in H2Odest in a septum flask and degas

– Succinate (1 M): Dissolve in ddH2O in a septum flask and degas

3.10.2 Methods

All enzymatic assays (Unden et al., 1980) were performed at 37 °C in 50 mM

phosphate buffer, pH 7.4 in anaerobized cuvettes (path length 0.4 cm) in

the presence of 150 μM DMN (Lancaster et al., 2005). Then 4–8 μL of



Wolinella succinogenes Membrane Protein Production



117



the enzyme (target concentration 5–12 μg/mL) is added and the samples

are incubated for 90 s prior to the measurement.

3.10.3 Measurement of the fumarate reduction activity (benzyl

viologen ! fumarate assay)

The fumarate reduction activity is measured using the artificial substrate

benzyl viologen. It is an assay measuring the fumarate reduction activity

of the two soluble subunits A and B independent of the membraneembedded C subunit. Benzyl viologen, a colorless substrate in the oxidized

form, is reduced by additions of small aliquots of dithionite forming a dark

violet color. As it binds artificially to the B subunit of the QFR, it serves as an

electron donor for reducing fumarate. The reaction is started with the addition of fumarate and the activity is monitored by measuring the change of

the absorbance at λ ¼ 546 nm (ε546 ¼ 19.5 mMÀ1 cmÀ1, d ¼ 0.4 cm).

1. Fill 980 μL of phosphate buffer (50 mM) pH 7.4 (or 50 mM Tris pH 8.0

in case of the SdhABE complex) in an anaerobic cuvette with a microliter syringe.

2. Add 10 μL of a benzyl viologen solution (0.1 M).

3. Reduce the compound by adding 2–5 μL of a sodium dithionite solution

(40 mg/mL); absorbance at λ ¼ 546 nm should reach a value of $1.4.

4. Add enzyme (5–12 μg/mL) and wait until the absorbance has stabilized.

5. Start the reaction with 10 μL of fumarate solution (1 M).

6. Calculate the initial slope of the spectrum after fumarate addition over

$10 s.

3.10.4 Measuring the quinol oxidation activity

(DMNH2 ! fumarate assay)

The assay of quinol oxidation by fumarate is performed according to Unden

and Kr€

oger (1981). The measurement is based on the quinol oxidation activity by simultaneous reduction of fumarate. In contrast to the fumarate reduction, the quinol oxidation is strictly dependent on the complete enzyme

including the C subunit. As a quinol is needed, the synthetic menaquinone

analogon DMN has to be pre-reduced by the addition of small aliquots of a

NaBH4 solution. After starting the reaction by addition of fumarate, the

reoxidation of DMNH2 is monitored spectrometrically by recording the

absorbance difference at λ ¼ 270 and 290 nm (ε270–290 ¼ 15.2 mMÀ1 cmÀ1,

d ¼ 0.4 cm).

1. Fill 975 μL of phosphate buffer (50 mM) pH 7.4 in an anaerobic cuvette

with a microliter syringe.



118



Michael Lafontaine and C. Roy D. Lancaster



2. Add 10 μL of a DMN solution (20 mM). The absorbance at λ ¼ 270 nm

should reach a value of $1.2. The absorbance at λ ¼ 290 nm should

remain constant during the complete recording.

3. Pre-reduce the quinone by adding 2.5–5 μL of a potassium borohydride

solution (20 mg/mL). The absorbance at λ ¼ 270 decreases to a value of

$0.3.

4. Add enzyme (5–12 μg/mL) and wait until the absorbance has stabilized.

5. Start the reaction with 10 μL of fumarate solution (0.1 M).

6. Calculate the initial slope of the difference spectra after fumarate addition

over $10 s.

3.10.5 Measuring the quinone reduction activity

The QFR as a SQOR is capable of reducing quinones by succinate oxidation. To measure the quinone reduction activity, the synthetic menaquinone analogon DMN is reduced by addition of succinate. The

procedure is basically the same as in 3.10.4 except that the quinone is not

pre-reduced by addition of potassium borohydride and succinate is used

instead of fumarate.

1. Fill 970 μL of phosphate buffer (50 mM) pH 7.4 in an anaerobic cuvette

with a microliter syringe.

2. Add 10 μL of a DMN solution (20 mM). The absorbance at λ ¼ 270 nm

should reach a value of $1.2. The absorbance at λ ¼ 290 nm should

remain constant during the complete recording.

3. Add enzyme (5–12 μg/mL) and wait until the absorbance has stabilized.

4. Start the reaction with 10 μL of succinate solution (1 M).

5. Calculate the initial slope of the difference spectra after succinate addition over $10 s.

3.10.6 Calculating of the relative catalytic activity

The relative catalytic activity is a useful measure for comparing protein

activities. It is the ratio of the specific quinol oxidation activity and the specific fumarate reduction activity. The quotient obtained for the wild-type or

reference protein is set to 100% allowing the comparison of RCA values of

variants independent of preparation quality.



ACKNOWLEDGMENTS

We thank all our collaborators as specified in our cited publications, in particular Hanno

Juhnke, Mauro Mileni, and J€

org Simon for their contributions. Support of our research

by the Deutsche Forschungsgemeinschaft (DFG, grants INST 256/275-1 FUGG and



Wolinella succinogenes Membrane Protein Production



119



256/299-1 FUGG, SFB 472-Lancaster, GK 845-Lancaster, and GK 1326-Lancaster), the

state of Saarland (grant LFFP 11/02), and Saarland University is gratefully acknowledged.



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CHAPTER SIX



Membrane Protein Expression

and Analysis in Yeast

Katrien Claes*,†,{, Mouna Guerfal*,†,{, Nico Callewaert*,†,{,1

*Unit of Medical Biotechnology, Department of Medical Protein Research, VIB-UGhent, Ghent, Belgium



Inflammation Research Center, VIB-UGhent, Ghent, Belgium

{

Department of Biochemistry and Microbiology, Laboratory for Protein Biochemistry and Biomolecular

Engineering, Ghent University, Ghent, Belgium

1

Corresponding author: e-mail address: nico.callewaert@vib-ugent.be



Contents

1. Theory

2. Equipment

3. Materials

3.1 Solutions and buffers

4. Protocol

4.1 Preparation

4.2 Duration

5. Step 1: Transformation of Y. lipolytica

5.1 Overview

5.2 Duration

5.3 Tip

5.4 Tip

5.5 Tip

6. Step 2: Small-Scale Membrane Protein Expression

6.1 Overview

6.2 Duration

6.3 Tip

6.4 Tip

7. Step 3: Membrane Protein Preparation

7.1 Overview

7.2 Duration

8. Step 4: Membrane Protein Analysis: Expression Levels

8.1 Overview

8.2 Duration

8.3 Tip

9. Step 5: Membrane Protein Analysis: Functionality

9.1 Overview

9.2 Duration

9.3 Tip



Methods in Enzymology, Volume 556

ISSN 0076-6879

http://dx.doi.org/10.1016/bs.mie.2014.12.010



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All rights reserved.



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