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Step 1: Transformation of Y. lipolytica

Step 1: Transformation of Y. lipolytica

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Step 1: Transformation of Yarrowia lipolytica

Preparation of competent cells:

1.1 Inoculate culture in YPD/citrate buffer overnight, shaking at 28 °C

1.2 Dilute the cells in YPD/citrate buffer and grow overnight, shaking at 28 °C, until

cultures reach an OD600 between 5 and 10

1.3 Centrifuge cells at 500×g, 4 °C, for 10 min

1.4 Wash the cells twice in TE-buffer

1.5 Resuspend the pellet in 0.1 M LiAc and incubate with gentle shaking (70 rpm) for

1 h at 28 °C

1.6 Centrifuge cells at 500×g, 4 °C, for 2 min

1.7 Resuspend the cells in 0.1M LiAc and keep on ice or flash freeze until further use

Tip: Cells can be stored at 4 °C up to 48 h

Transformation of competent cells:

1.8 In a sterile tube, add carrier DNA and the expression plasmid to the competent

cells

1.9 Tap the tube gently and put it without shaking at 28 °C for 15 min

1.10 Add the PEG-4000 solution very gently to the tube

Tip: It is best to pipette the solution against the wall of the tube while

slightly rotating it, to ensure the solution flows underneath the cells

1.11 Incubate the cells for 1 h at 28 °C with shaking, 200 rpm

1.12 Heat shock the cells for 10 min in a warm water bath at 39 °C and then transfer

them to ice

1.13 Add LiAc, mix gently, and plate the cells on correct selective plates

1.14 Incubate the plates for 2 days at 28 °C until colonies become visible

1.15 Streak single colonies to a new selective plate and incubate again for 2 days,

keep this plate as the master plate



Figure 2 Detailed flowchart of step 1.



Preparation of competent cells

1.1 Inoculate a 10-ml culture in YPD/citrate buffer in a shaking incubator

at 28 °C overnight.

1.2 Dilute the cells in 25 ml YPD/citrate buffer in a 125-ml baffled shake

flask so they reach an OD600 of between 5 and 10 the next morning

(see Notes 1 and 2).

Note 1: 1 OD600 ¼ 1.2 Â 107 cells/ml.

Note 2: The generation time of Y. lipolytica PO1d is about 2 h.



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Centrifuge the culture for 10 min, 500 Â g, 4 °C.

Wash the cells twice in 10 ml of TE-buffer.

Resuspend the cell pellet in 20 ml of 0.1 M LiAc and incubate them

for 1 h at 28 °C with gentle shaking (70 rpm).

1.6 Centrifuge the cells for 2 min, 500 Â g, 4 °C.

1.7 Resuspend the cells in 1 ml of 0.1 M LiAc and keep them on ice until

further use (Tip 5.3).

Transformation of competent Y. lipolytica cells

1.8 In a sterile 15-ml tube, add 2.5 μl of 10 mg/ml of carrier DNA,

200–500 ng of plasmid DNA, and 100 μl of competent cell

suspension.

1.9 Tap the tube gently and put it for 15 min at 28 °C, without shaking.

1.10 Very slowly add 700 μl of PEG-4000 solution to the tube (Tip 5.4).

1.11 Incubate the tube for 1 h at 28 °C, 200 rpm.

1.12 Heat shock the cells for 10 min at 39 °C in a warm water bath and

then transfer to ice.

1.13 Add 1.2 ml of 0.1 M LiAc, mix gently, and plate different amounts of

cells on the UraÀ selective plates (e.g., 100 and 200 μl of the transformation suspension).

1.14 Incubate the plates for 2 days at 28 °C until colonies are clearly visible.

1.15 Streak single colonies again to a new selective plate to ensure picking

of true single colonies in the following steps. Incubate this plate again

for about 2 days at 28 °C and keep this as the master plate (Tip 5.5).

1.3

1.4

1.5



5.3 Tip

The competent cells can be stored at 4 °C for 48 h.



5.4 Tip

It is best to pipette the PEG-4000 solution against the wall of the tube, while

turning the tube slightly. This ensures that the PEG-4000 solution flows

underneath the cells, giving better transformation results.



5.5 Tip

The obtained strains can be preserved at À80 °C for long-term storage. To

this end, a 2-ml liquid culture is grown in YPD at 28 °C, 200 rpm, overnight. The next day, mix 300 μl of 100% glycerol with 700 μl of cell suspension in a cryo-vial. Immediately submerse the vial in liquid nitrogen and

then transfer it to a À80 °C freezer.



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6. STEP 2: SMALL-SCALE MEMBRANE PROTEIN

EXPRESSION

6.1 Overview

Insertion of the expression plasmid in the genome of Y. lipolytica can be

determined by PCR on genomic DNA of the transformed strain. Standard

protocols for the isolation of genomic DNA from yeast and consecutive

PCR can be used (e.g., MasterPure Yeast DNA purification kit, EpiCentre).

In case the copy number of the integrated plasmid is of interest, Southern

blotting can be performed. Here as well, standard protocols can be used

(e.g., DIG High prime DNA labeling and detection starter kit II, Roche).

To analyze the expression of the membrane protein of interest, a smallscale cultivation can be set up (Fig. 3). Because expression levels are highly

influenced by the location of integration and the copy number, it is recommended to screen at least 12 clones. This step describes the induction

of membrane protein expression in a 24-deep-well format.



6.2 Duration

2–3 Days

Step 2: Small-scale membrane protein expression screening

2.1 Inoculate 12–24 single colonies in YTG in a 24-well plate and seal with AirPore

tape

2.2 Incubate the plate at 28 °C for 24 h in a shaking incubator, 225 rpm

2.3 Centrifuge the plate at 500×g, room temperature, for 2 min

Tip: If you do not have a deep-well compatible rotor to your disposal,

transfer the cell suspensions to individual eppendorfs and then, after the final

centrifugation step, transfer the cell suspension back to the deep-well plate

2.4 Discard the medium and wash the cell pellet with 1× DPBS

2.5 Resuspend the cells in YTO to start membrane protein expression and incubate at

28 °C for another 24 – 48 h while shaking, 225 rpm

Tip: The time point at which maximum accumulation levels are observed

depends on the particular membrane protein. It is recommended to perform an

experiment in which the 16 – 96 h induction range is explored

2.6 Centrifuge the plate at 500×g, 4 °C, for 2 min

2.7 Wash the pellet twice with 1× DPBS to remove excess oleic acid



Figure 3 Detailed flowchart of step 2.



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133



2.1 Inoculate a single clone from the master plate (obtained in step 1) in

2 ml YTG in one well of a deep, round-bottom 24-well plate. Repeat

this step for 12–24 clones and seal the plate with AirPore tape.

2.2 Incubate the plate for 24 h at 28 °C in a shaking incubator, 225 rpm.

2.3 Centrifuge the plate at 500 Â g for 2 min at room temperature (Tip 6.3).

2.4 Discard the medium and wash the pellet with 1 Â DPBS.

2.5 Resuspend the pellet in 2 ml of YTO to induce membrane protein

expression and incubate for another 24–48 h at 28 °C in a shaking incubator, 225 rpm (Tip 6.4).

2.6 Harvest the cells by centrifugation at 500 Â g for 2 min at room

temperature.

2.7 Wash the pellet twice with 1 Â DPBS to remove excess oleic acid and

proceed to step 3 for expression analysis.



6.3 Tip

If you do not have a deep-well compatible rotor to your disposal, you can

transfer the cell suspensions to individual 2-ml Eppendorf tubes and then,

after the final centrifugation step, transfer the cell suspensions back to the

deep-well plate.



6.4 Tip

The time point at which maximum accumulation levels are observed

depends on the particular membrane protein. We recommend 48 h in a pilot

experiment, followed by a second experiment in which the range of 16–96 h

is explored. Usually, an optimal expression level is observed between

24 and 48 h.



7. STEP 3: MEMBRANE PROTEIN PREPARATION

7.1 Overview

In this step, we provide details on the isolation of membrane proteins from

Y. lipolytica, however, the protocol can be used for other yeast species as well,

e.g., P. pastoris (Fig. 4).



7.2 Duration

2h

3.1 Resuspend the cells obtained from step 2.6 in 1 ml of ice-cold DPBSPI buffer, transfer to a 2-ml screw-cap tube, and add the content of one



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Step 3: Membrane protein preparation from a cell culture

3.1 Resuspend the cells from step 2.6 in ice-cold DPBS-PI buffer, transfer to a screwcap tube and add a PCR tube full of glass beads

3.2 Break the cells upon vortexing using a Retsch mixer mill for 8 min at 4 °C

3.3 Centrifuge the cells at 300×g, 4 °C, for 5 min

3.4 Transfer the supernatant to a new Eppendorftube and centrifuge at 16,000×g, 4 °C,

for 45 min

3.5 Add ice-cold DPBS-PI buffer to the pellet and resuspend using sonication (20 s

ON, 37% amplitude), on ice. Use the same day or flash freeze and store at

−80 °C.



Figure 4 Detailed flowchart of step 3.



3.2

3.3

3.4



3.5



300-μl PCR tube full of glass beads to enable mechanical rupture of the

cell wall.

Break the cells through vigorous vortexing using a Retsch mixer mill

for 8 min at 4 °C.

Centrifuge the samples at 300 Â g for 5 min at 4 °C to remove unbroken cells, cell wall material, and glass beads.

Transfer the supernatant to a new Eppendorf tube and centrifuge at

16,000 Â g for 45 min at 4 °C to pellet the membrane fraction containing the membrane proteins.

Add 500 μl of ice-cold DPBS-PI buffer to the pellets and resuspend

using sonication: 20 s at 37% amplitude in an ice bath (VCX 500

sonicator with 1/8" microtip). Store the obtained membrane protein

preparations on ice when used the same day, otherwise flash freeze

in liquid nitrogen and store at À80 °C until further use.



8. STEP 4: MEMBRANE PROTEIN ANALYSIS: EXPRESSION

LEVELS

8.1 Overview

In this step, we describe how the isolated membrane proteins are analyzed

using Western blotting (Fig. 5). In order to be able to compare expression

levels from the different clones, a normalization is performed. This can be

done by starting the membrane protein extraction from identical cell densities. However, when the extraction efficiency is not identical for all samples, this will result in errors. So it is best to delay the normalization until the

very end. Therefore, we determine the total protein concentration of

the membrane samples after extraction. Because the fraction of the



Membrane Protein Expression and Analysis in Yeast



135



Step 4: Quantitative membrane protein analysis using Western

blotting

4.1 Determine the total membrane protein concentration using the BCA protein assay

reagent kit

4.2 Prepare 10 µg total membrane protein for each sample, preferably in a total of 20 µl

DPBS-PI buffer and add 5 µl of 5× Laemmli loading dye

4.3 Analyze the samples by SDS-PAGE and Western blotting using the appropriate

primary and secondary antibodies

Tip: Do not boil protein samples before loading them on gel to prevent

protein aggregation. In stead, load the samples immediately or heat them for 5–10 min

at maximum 50 °C



Figure 5 Detailed flowchart of step 4.



overexpressed membrane protein of interest is usually still small compared to

the endogenous membrane protein content, the total protein concentration

can be used as a calibrant.



8.2 Duration

1 Day

4.1 Determine the total protein concentration, e.g., BCA protein assay kit

(Pierce).

4.2 Prepare 10 μg of total membrane protein for each sample, preferably in

20 μl of DPBS-PI buffer, and add 5 μl of 5 Â Laemmli loading dye.

4.3 Analyze the samples by SDS-PAGE and Western blotting using appropriate primary and secondary antibodies.



8.3 Tip

When preparing the samples for SDS-PAGE, it is essential not to boil the

samples to prevent membrane protein aggregation. The samples can be

loaded on SDS-PAGE immediately after the addition of the Laemmli loading dye or, when preferred, incubated for 5–10 min at maximum 50 °C.

Some exploration may be required to obtain optimal results.



9. STEP 5: MEMBRANE PROTEIN ANALYSIS:

FUNCTIONALITY

9.1 Overview

Of course, the type of functional assay will entirely depend on the type of

membrane protein under study. Indeed, for many membrane proteins no

readily available assay may exist, severely complicating the work with such



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proteins. Here, we describe how to perform radioligand binding assays for

G-protein-coupled receptors in order to determine whether the overexpressed protein is in a ligand-binding competent folding state. Since

the use of radioligand binding requires radiolabeled molecules, this technique is limited to proteins for which these compounds are available. The

protocol is based on the one described in Fraser (2006).

In the first part, we describe a more elaborate way of preparing membrane proteins, which includes solubilization of the membrane proteins.

The second part explains how to perform the binding assay itself. The assay

is performed at least in duplicate and necessary controls are included, such as

no radioligand, no membrane protein and determination of nonspecific

binding of the radioligand by competition with a nonradioactive (cold)

ligand. In case of the adenosine A2A receptor, we use an excess amount

of theophylline as cold ligand. For some membrane proteins, a natural interactor can also still be present in the membrane preparation. In our case, we

add adenosine deaminase to the membrane protein preparation. This results

in the deamination of adenosine, thereby preventing further interaction of

the molecule with the receptor. The protocol described below is specifically

developed for the analysis of the aforementioned G-protein-coupled receptor. It should be taken into account that this method can serve as a basis to

perform radioligand binding assays, but it is very likely that for each other

membrane protein, modifications will be required.

All the steps described below need to be performed on ice as much as

possible. For the flowchart, see Fig. 6.



9.2 Duration

2 Days starting from a cell pellet in which the membrane protein of interest is

produced.

Membrane protein preparation for radioligand binding assay

5.1 Resuspend a 2-ml cell pellet, containing cells expressing the membrane protein of interest, in ice-cold DPBS-PI buffer, transfer to a

screw-cap tube, and add the content of one PCR tube full of glass

beads to enable mechanical rupture of the cell wall.

5.2 Break the cells through vigorous vortexing using a Retsch mixer mill

for five times 2 min at 4 °C, allowing the samples to cool down again

for 1 min in between each cycle.

5.3 Centrifuge the samples at 1000 Â g for 30 min at 4 °C to remove

unbroken cells, cell wall material, and glass beads.



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Membrane Protein Expression and Analysis in Yeast



Transfer the supernatant to an ultracentrifuge tube (e.g., polycarbonate

tubes, Beckman Coulter) and centrifuge at 100,000 Â g for 1 h at 4 °C.

5.5 Remove the supernatant and add 500 μl of ice-cold DPBS-PI buffer

to the pellet.

5.6 Resuspend the sample using sonication: 2 s ON, 1 s OFF for a total of

20 s ON, at 37% amplitude in an ice bath (VCX 500 sonicator with

1/8" microtip).

5.7 Solubilize the membrane proteins by putting them on an end-overend rotator at 4 °C in the presence of 1% DDM for 1 h (Tip 9.3).

5.8 Transfer the solution to a new ultracentrifuge tube and centrifuge at

200,000 Â g for 1 h at 4 °C.

5.9 Transfer the supernatant to a new Eppendorf tube and store the

obtained membrane protein preparations on ice when used the same

day, otherwise flash freeze and store at À80 °C until further use.

Radioligand binding assay

5.10 Determine the total membrane protein concentration using a

BCA assay.



5.4



Step 5: Functional analysis of the membrane protein sample using a

radioligand binding assay

Membrane protein preparation for radioligand binding assay:

5.1 Resuspend a cell pellet from step 2.6 in DPBS-PI buffer, transfer to a screw-cap

tube and add a PCR tube full of glass beads

5.2 Break the cells upon vortexing using a Retsch mixer mill for five times 2 min at 4 °C

5.3 Centrifuge the cells at 1000×g, 4 °C, for 30 min

5.4 Transfer the supernatant to an ultracentrifuge tube and centrifuge at 100,000×g,

4 °C, for 1 h

5.5 Add ice-cold DPBS-PI buffer to the pellet

5.6 Resuspend the pellet using sonication (2 s ON, 1 s OFF, for a total of 20 s

ON, 37% amplitude), on ice.

5.7 Solubilize the membrane proteins by adding 1% DDM for 1 h on an end-overend rotator

Tip: DDM is often used as a first-in-line detergent, but to obtain the highest

amount of solubilization, a detergent screen should be performed

5.8 Transfer the solution to a new ultracentrifuge tube and centrifuge at 200,000×g,

4 °C, for 1 h



Figure 6 Detailed flowchart of step 5.

(Continued)



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5.9 Transfer the supernatant to a new Eppendorf tube and store the obtained

membrane protein preparations on ice when used the same day, otherwise flash freeze

and store at −80 °C until further use

Radioligand binding assay:

5.10 Determine the total membrane protein concentration using the BCA protein

assay reagent kit

5.11 Prepare radioligand dilution series and dilute the cold ligand

5.12 Prepare the membrane protein solution in binding buffer and supplement when

necessary

5.13 Add cold ligand or an equal volume of binding buffer to a FACS tube

5.14 Add the membrane protein sample

and the radioligand to the tube, mix and

incubate for 1 h at room temperature



5.15 Meanwhile activate the

Whatman glass fiber filters with

0.1% polyethylenimine



5.16 Assemble the harvester by putting the filter in place and fill up the buffer tank

with binding buffer. Allow a few milliliters of buffer to flow through the filter

5.17 Harvest the samples on the filter and wash with a few milliliters of binding

buffer. Purge to remove any excess drops of buffer

5.18 Take the filter out and cut out the spots on which the membrane proteins have

bound. Put the filters in separate tubes

5.19 Add scintillation fluid and store overnight at 4 °C

5.20 Put the tubes in the liquid scintillation counter to generate the data

5.21 Determine the binding affinities based on the obtained data using rectangular

hyperbole curve fitting



Figure 6—Cont'd



5.11 Prepare radioligand dilution series (e.g., 0.05–12 nM final radioligand

concentration) and dilute the cold ligand to test for nonspecific binding (e.g., 10 mM final concentration).

5.12 Prepare the membrane protein solution at a concentration of

25 μg/ml in binding buffer supplemented with, e.g., adenosine

deaminase.

5.13 Add 50 μl of cold ligand (control samples) or 50 μl of binding buffer to

a FACS tube.

5.14 Add 400 μl of membrane protein sample and 50 μl of radioligand at

the desired concentration to the tube. Mix and incubate for 1 h at

room temperature.



Membrane Protein Expression and Analysis in Yeast



139



5.15 In the meantime, activate the Whatman glass fiber filters by putting

them in 0.1% polyethylenimine.

5.16 Assemble the harvester by putting the activated filter in place and fill

up the buffer tank with binding buffer. Turn on the pump and allow a

few milliliters to flow through the filter.

5.17 After an hour, harvest the samples and wash the filter twice with a few

milliliters of binding buffer. Purge to remove any excess drops

of buffer.

5.18 Take the filter out of the harvester and cut out the spots on which the

membrane protein has bound. Put the filters in separate tubes.

5.19 Add 3 ml of scintillation fluid and store overnight at 4 °C.

5.20 Put the tubes in the liquid scintillation counter to quantify the radioactivity. The result will be a list of disintegrations per minute for each

sample.

5.21 Binding affinities of the radioligand can now be determined upon

rectangular hyperbole curve fitting using, e.g., KaleidaGraph software. The hyperbole will approach the maximal specific binding

(Bmax) asymptotically and can then be expressed as nanomolar or picomolar per milligram of total membrane protein.



9.3 Tip

For the solubilization of membrane proteins, many different detergents are

available. DDM is often used as a first-in-line detergent, but in order to

obtain the highest amount of solubilized membrane protein in a functional

state, a detergent screening should be performed.



REFERENCES

Barth, G., & Gaillardin, C. (1997). Physiology and genetics of the dimorphic fungus Yarrowia

lipolytica. FEMS Microbiology Reviews, 19, 219–237.

Bonazzi, M., Lecuit, M., & Cossart, P. (2009). Listeria monocytogenes internalin and

E-cadherin: From bench to bedside. Cold Spring Harbor Perspectives in Biology, 1(4),

a003087.

Fraser, N. J. (2006). Expression and functional purification of a glycosylation deficient version

of the human adenosine 2a receptor for structural studies. Protein Expression and Purification, 49, 129–137.

Guerfal, M., Claes, K., Knittelfelder, O., De Rycke, R., Kohlwein, S. D., & Callewaert, N.

(2013). Enhanced membrane protein expression by engineering increased intracellular

membrane production. Microbial Cell Factories, 12, 122.

Hodges, R. S., Heaton, R. J., Parker, J. M. R., Molday, L., & Molday, R. S. (1988).

Antigen-antibody interaction: Synthetic peptides define linear antigenic determinants

recognized by monoclonal antibodies directed to the cytoplasmic carboxyl terminus

of rhodopsin. The Journal of Biological Chemistry, 263, 11768–11775.



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Sieben, C., Kappel, C., Zhu, R., Wozniak, A., Rankl, C., Hinterdorfer, P., et al. (2012).

Influenza virus binds its host cell using multiple dynamic interactions. Proceedings of the

National Academy of Sciences of the United States of America, 109(34), 13626–13631.

van Meer, G., Voelker, D. R., & Feigenson, G. W. (2008). Membrane lipids: Where they are

and how they behave. Nature Reviews Molecular Cell Biology, 9, 112–124.



FURTHER READING

Guerfal, M., Ryckaert, S., Jacobs, P. P., Ameloot, P., Van Craenenbroeck, K., De

Rycke, R., et al. (2010). The HAC1 gene from Pichia pastoris: Characterization and effect

of its overexpression on the production of secreted, surface displayed and membrane proteins. Microbial Cell Factories, 9, 49.



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