terminal StrepII-StrepII-HA (SSH) and cMyc tag variants

Facing the stability and expression problems using the FLAG tag it was decided to replace the conserved endogenous gene coding for TbAMPKβ with constructs containing the tandem SSH tag and the selectable marker puroR flanked by the 3’UTR of the target gene (Figure 25). In contrast, the TbAMPKγ subunit was modified to be expressed with a C-terminal triple cMyc tag using hygroR as selectable marker. This would allow the affinity purification/immunoprecipitation with two different matrices by the construction of a strain expressing both tagged variants at the same time (co-transfections).

Figure 25 Second series of targetting constructs for homologous recombination of TbAMPKβ and TbAMPKγ with SSH-tag and cMyc-tag respectively. For cloning reasons, the SalI restriction enzyme sequence present between the tag and the α-β-tubulin intergenic region was replaced by a NcoI restriction enzyme sequence.

Plasmid construction : The replacement of the tag for the TbAMPKβ construct was designed as outlined above, obtained by gene synthesis (GENEWIZ inc., USA) and subsequently ligated into the MCS of pUC57 using the XbaI and HindIII restriction sites (Figure 25 and Table 6). The sequence between XbaI and HindIII, containing the end of the gene sequence, the SSH tag and the α-β-tubulin intergenic region (obtained by gene synthesis, see table 1 appendix A) was inserted into the previously ordered plasmid, pUC57_Nysm-TbAMPKβ-SSF, (Figure 25 and Table 5) between XbaI and HindIII restriction site. The plasmid, pUC57_Nysm-TbAMPKβ-SSH, was verified by automated sequencing (Microsynth AG, CH)

The triple cMyc tag for the TbAMPKγ construct was obtained by PCR amplification of the region of interest from the plasmid pMOTag43M-crelox-HYG (a gift from George

Cross, Addgene plasmid # 24031). The sequence was cloned between XhoI and NcoI of the previously cloned pUC57_Nysm-TbAMPKγ-SSH plasmid harbouring puroR. All primers used for cloning are shown in Table 6. The final plasmid, pUC57_Nysm-TbAMPKγ-cMyc was purified and co-digested with XbaI and BamHI to isolate the sequence of interest.

Table 6 Primers sequences for endogenous tagging of TbAMPKγ

Primers Sequence* PCR condition and

DNA source

*XbaI, XhoI, NcoI, HindIII, NotI, BamHI X°C : annealing temperature Y : extension time

2A.2.6. Stable transformation of T. brucei and PCR validation

Stable transfomation of BSF was performed using a non-viral nucleofection method.

The efficiency of transfection using program X-001 of AMAXA Nucleofector® II (Lonza, Switzerland) was in the range of 1.3-2.0 x 10^-4 (several orders of magnitude lower compared to the efficiency for procyclic forms). The buffer used for transfection was an home-made T. brucei transfection buffer (Tb-BSF buffer) (50 mM HEPES, 90 mM sodium phosphate, 5 mM potassium chloride, 0.15 mM calcium chloride, pH 7.3) recently described by Burkard et al. [22, 23].

The digestion products (linearized or double digested) were ethanol precipitated, resuspended in sterile water and used for electroporation to replace TbAMPKβ or TbAMPKγ alleles in Nysm T. brucei by homologous recombination. The final concentration was determined with a Nanodrop device (NanoDrop™

One/One^C Microvolume UV-Vis Spectrophotometer, Thermofisher Scientific, MA USA).

6×10⁷ cells in log phase were centrifuged 10 min at 600 x g at RT. The pellet was resuspended in 100 μL Tb-BSF buffer and mixed with 10 μg of purified linearized plasmid or 3 μg of the sequence obtained by double digestion. Electroporation was

performed using 2 mm gap cuvettes (BTX, Harvard apparatus) with program X-001 of the Amaxa Nucleofector. Immediately after transfection, the cells were transferred to 25 mL HMI-9 supplemented with 10% FCS. Dilutions of 1:100, 1:500 or 1:1000 were performed and 1 mL/well was distributed into 24-well plates. After 22 h of incubation, the antibiotic was added for selection of positive recombinant clones. Five to ten days post transfection resistant clones were transferred to culture flasks while maintaining antibiotic pressure. The plasmids used as well as the concentration of antibiotic used for the selection of the different strains are shown in Table 7.

Table 7 Antibiotics used for selection of positives clones.

Plasmid or PCR construct Antibiotic used for final selection

gDNA was extracted from the positive clones and the correct integration was verified by PCR using actin as positive control.

For the verification of the endogenously tagged subunits, the forward primers were engineered to anneal to an area close to the middle of the TbAMPKβ (or TbAMPKγ) gene whereas the reverse primer was designed either to anneal the α/β tubulin region (present between the resistance cassette and the tag) or to anneal downstream of the 3’UTR region used for the homologous recombination. Primers used are shown in Table 8.

Table 8 Primers and their respectively sequence used for the validation of the correct insertion.

Primer Sequence

2A.2.7. Affinity purification/immunoprecipitation of the intracellular heterotrimeric complex formed with tagged TbAMPKβ or TbAMPKγ and subsequent identification of TbAMPKα subunit

TbAMPKβ-SSH tag variant : Total native protein extraction was carried out starting from 2x10⁸ parasite cells. After washing in 10 mL PBS/Glucose (addition of 1 mg/mL Glucose) solution the pellet was suspended in 200 μL lysis buffer (30 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 2 mM DTT, Protease Inhibitors cocktail, pH7.4) and sonicated for 10 s (10% power) with SONOPULS™ Digital Ultrasonic Homogenizers (Bandelin, D). The supernatant was collected after centrifugation at 20’000 x g (30 min at 4°C). Protein concentration was measured using Bradford protein assay using BSA as a standard. An equivalent of 0.6 mg of total protein extract was incubated with anti-HA resin (Pierce Anti-anti-HA agarose, ThermoScientific) for 4 h on a rocking platform at 4°C. Three washing steps, consisting of an incubation of 5 min with 200 μL of lysis buffer and 10 s centrifugation at 12'000 x g were performed. Finally, the resin was incubated with 30 μL of 4xLaemmli loading buffer (4xSD: 220 mM Tris-Base, 7.3%

SDS, 36% Glycerol, 200 mM β-Mercaptoethanol, 0.01% bromophenol blue), heated (3 min at 95°C), analyzed by SDS-PAGE and developed by silver stain (Invitrogen Life Technologies) following manufacturer’s protocol. As an alternative, the bound proteins were eluted with HA-peptide in the final elution step. HA-peptide elution was performed 3 times by applying 1 bed volume of Pierce HA peptide (1 mg/mL) to the column and incubated 15 min at 30°C. The eluate was collected after 10 s centrifugation at 12'000 x g.

TbAMPKγ-cMyc tag variant : Immunoprecipitation using the TbAMPKγ-cMyc tagged variant was carried out using the similar protocol outlined before. Briefly, total protein extracts were prepared from 2x10⁸ cells. After washing in PBS/Glucose solution the pellet was suspended in 1 mL lysis buffer and sonicated 10 s (10% power). The clarified supernatant was collected and incubated for 3 h with 70 μL of EZview red anti-cMyc affinity gel (Sigma-Aldrich), previously equilibrated in lysis buffer, on a rocking platform at 4°C. Then the gel was washed several times before adding 60 μL 2xSB (3 min at 95°C) and subsequent SDS-PAGE separation and detection of protein bands with silver staining (Invitrogen Life Technologies).

LC/MS/MS analysis: LC-MS analysis were performed after cutting bands of interest in SDS-PAGE gel silver stained and send to the Protein Analysis Group of the Functional Genomics Center in Zurich.

2A.2.8. Western blot

Sample preparation : 1-5 x 10⁷ T. brucei cells were centrifugated at 600 g for 10 min at 4°C and the pellet was washed in 10 mL PBS/Glucose and 1mM phenylmethylsulfonyl fluoride (PMSF) and cetrifuged again at 600 x g for 10 min at 4°C. The pellet was resuspended in 1 mL PBS/Glucose, transferred into a new 1.7 mL tube, and centrifuged again. The pellet was resuspended in 50 µL 2xSD buffer to obtain a final concentration of 2x10⁵ cells/µL. Finally, the samples were boiled at 95°C for 5 min and charged on SDS-PAGE gel or stored at -20°C.

SDS page gels : If not stated otherwise in the individual chapters the standard gels used were 12% polyacrylamide gels (Table 9).

Table 9 Composition of 12% polyacrylamide gels

Components Separating Gel [μL] Stacking gel [μL]

30% Acrylamide solution 4000 780

1.5 M Tris-HCl, pH 8.8 2500 - mA for 45 min to separate the proteins by their size in running buffer (Tris 25mM, 191.6 mM Glycin, 0.1% SDS). The size marker used for Western Blot analysis was a PageRuler^TM prestained protein ladder (10 to 180kDa, ThermoFisher Scientific, MA USA), whereas for Coomassie staining gels a PageRuler^TM unstained protein ladder (10 to 200kDa, ThermoFisher Scientific, MA USA) was used.

TbBip (homologous to immunoglobulin heavy chain binding protein), a protein of 70kDa is the housekeeping gene of reference used in all experiments as loading control.

Transfer and blocking : The obtained gel and two thick Whatman papers (PROTEAN Extra thick Blot paper from BIO-RAD) were equilibrated in cold (4°C) transfer buffer (25mM Tris, 191.6 mM Glycin, 0.1% SDS, 20% methanol) for 30 min while shaking.

The nitrocellulose membrane (0.22μM or 0.45μM pore size from BIO-RAD, USA) was equilibrate in ddH2O under the same conditions. The transfer was performed with Trans-blotR SD semi-dry transfer system from BIO-RAD for 40 min at 15 V, 260 mA.

After the transfer the membrane was washed twice with ddH2O before incubation with blocking solution of 3% BSA in TBS-T (25 mM Tris, 65 mM NaCl, 2.7 mM KCl, 0.05%

Tween20, pH 7.4) for 1h30 at RT on a shaking platform. In some cases 3% Low Fat Milk powder was used to replace BSA.

To verify the efficacy of the transfer, if necessary, the gel was Coomassie blue-stained and the membrane was stained with Ponceau S staining solution (Sigma-Aldrich).

Antibody incubation and detection systems : After blocking, the membrane was directly incubated with the first antibody solution (diluted in blocking solution) o/n at 4°C (if not stated otherwise) on a rocking platform.

Before incubating with the secondary antibody the membrane was washed 4 times for 5-10 min in 10 mL of TBS-T on a rocking platform.

Secondary antibody was either conjugated to Horse-radish peroxidase (HRP) or to a fluorescent dye. In both cases dilution was made in blocking solution and incubation at RT for 1h30 on a rocking platform.

The detection system is dependent on the secondary antibody used. The HRP-conjugated antibody was detected by chemiluminescence using ECL Western blotting reagents (Amersham Bioscences), the membrane was incubated for 3 min with the ECL buffer and was developped on a RX film with a AGfA Curix60 Film processor.

When the fluorescend dye was used the membrane was directly analyzed with a Odyssey scanner (LI-COR). Table 10 summarizes the antibodies (primary and secondary) used in this work.

Table 10 List of primary and secondary antibodies.

His-tag mouse anti-His, (1 :2'500 dilution, QIAGEN #34660)

2A.3. Results and discussion

2A.3.1. Replacement of endogenous TbAMPKβ and TbAMPKγ genes by a tagged variant

For isolation and subsequent identification of all members of the parasite AMPK by affinity/immunoprecipitation, it was mandatory to create genetically modified T. brucei lines expressing tagged variants of the TbAMPKβ (Tb927.8.2450) and TbAMPKγ (Tb927.10.3700) subunits. It was decided to replace endogenous genes to maintain control of expression by native promoters and not to induce overexpression. Indeed, the toxic effect due to the accumulation of proteins may result in a loss of sensitivity during the downstream isolation processes of the AMPK complex. In addition, the decision to tag the protein of interest at the C-terminus (TbAMPKβ and TbAMPKγ) was based on recently published observations regarding the mammalian AMPK, showing that tagging the C-terminus would not interfere with heterotrimeric complex formation [8]. The initial project consisted in labelling the protein of interest with a double tag, as required by the TAP tagging system [24], thus allowing a two-step purification protocol.

For this project, the original label used in the TAP-tag system was replaced by the SSF tag and, at a later stage, by a SSH tag or a cMyc tag due to difficulties in detecting the protein labelled with the initial system. The construction required for homologous recombination was designed as follows: the tag, the α-β-tubulin intergenic region and the resistance sequence were flanked by two sequences of about 200 base pairs (bp) corresponding to the end of the gene of interest and the beginning of the 3'-untranslated region (UTR). For stable transfection, either the linearized plasmid or the double digested sequence containing only the sequence of interest was electroporated.

TbAMPKβ: StrepII-StrepII-FLAG tagged variant (Nysm-β-SSF)

The construct needed for homologous recombination containing a tandem SSF tag and puromycine resistance was synthetized and inserted into the multiple cloning site of the pUC57 plasmid, providing pUC57_Nysm-TbAMPKβ-SSF. Subsequent transfection into T. brucei provided resistant clonal cultures.

The presence of the insertion was verified by PCR for several clones (A-E) using primers between the TbAMPKβ gene and the α-β tubulin intergenic region. In addition, amplification between the TbAMPKβ gene and a 3'UTR sequence after the 200 bp region used for homologous recombination showed that the tagged protein sequence had indeed replaced the endogenous gene (Figure 26).

Figure 26 Validation of AMPKβ-StrepII-StrepII-FLAG tag insertion for clones A-E via PCR using gDNA. PCR internal to the tag inserted, between the β gene and the tubulin. PCR checking the correct insertion of the tag, between the β gene and the 3'UTR region. MWM: Molecular Weight Marker. Nysm: New York single marker cell line. Nysm-β-SSF (A-E): Nysm with TbAMPKβ subunit tagged in C-terminus with StrepII-StrepII-FLAG tag.

The presence of the AMPKβ-SSF tagged protein was verified by dot blot (Figure 27) and Western blot (Figure 28) analyses with anti-FLAG antibody. However, this antibody could not unambiguously demonstrate the presence of the expressed tagged protein in the resistant clones. The dot blot yielded a strong signal for all parasites extracts, including the negative control cell line T. brucei (Nysm), and also of the TbAK control protein which lacks a FLAG-tag.

Figure 27 Dot blot analyses of TbAMPKβ-StrepII-StrepII-FLAG expression. Different blocking solution were tested : low-fat milk powder and BSA. TbAK: T. brucei adenosine kinase. Nysm: New York single marker cell line. Nysm-β-SSF (clone A, B and E): Nysm with TbAMPKβ subunit tagged in C-terminus with StrepII-StrepII-FLAG tag.

Further Western blot analysis confirmed this finding. Only a single protein of around 50 kDa showed a strong signal with anti-FLAG antibody in both the clones and the negative controls. No specific signal was detected at 40 kDa, which would correspond to the expected size of the β tagged protein. These results let us conclude that the anti-FLAG antibody is not specific enough for our purposes. As previous dot blot analyses showed milk powder to be the best blocking agent compared to BSA, the western blot membrane was blocked with 5% low fat milk solution.

Figure 28 Western blot analyses of TbAMPKβ-SSF expression with FLAG Ab. Presence of a band of 50kDa in all samples, even negative controls, is shown in figure (A). TbBip (loading control) (B). TbAK: Trypanosoma brucei Adenosine kinase. Nysm: New York single marker cell line. Nysm-β-SSF (clone A, B and E): Nysm with TbAMPKβ subunit tagged in C-terminus with StrepII-StrepII-FLAG tag.

The detection of the StrepII double tag has been tested in all clones. However, the use of a specific kit for the detection of StrepII also failed to detect TbAMPKβ-StrepII-StrepII tagged protein in crude trypansome extracts. The only bands detected were those corresponding to the positive control, which was the molecular marker itself (composed of StrepII tagged fusion proteins (data not shown)). Since none of the tested conditions allow the detection of AMPK, we decided not to push forward the test of these clones.

Replacement of endogenous TbAMPKβ by StrepII-StrepII-HA tagged variant (Nysm-β-SSH)

It was decided to replace the FLAG tag with a triple HA tag, thus the final construct needed for homologous recombination containing a SSH tandem tag and a

puromycine resistance was cloned. After XbaI/BamHI digestion, ethanol precipitation and transfection in Nysm cell line, resistant clonal cultures were selected. The correct allele replacement was verified by PCR by amplification of the region between half of the gene and a 3'UTR sequence after the 200 bp region used for homologous recombination (Figure 29).

Figure 29 Validation of TbAMPKβ-SSH tag insetion for clone 1 via PCR from gDNA. Actin gene was used as control.

Nysm: New York single marker cell line. Nysm-β-SSH (clone 1): Nysm with TbAMPKβ subunit tagged in C-terminus with StrepII-StrepII-HA tag.

The presence of the HA-tagged protein was confirmed by western blotting using an antibody against the HA epitope. The crude protein extract equivalent to 1, 2 and 4 million of T. brucei cells was charged. In each lane a strong signal corresponding to the HA-tagged TbAMPKβ protein at 40 kDa was detected. The positive control consists of a protein extract of the D299V cell line (kindly provided by Dr. Pratricia Graven) overexpressing a HA-tagged Adenosine kinase of 37kDa size (Figure 30).

Figure 30 Verification of TbAMPKβ-StrepII-StrepII-HA expression in clone 1 using HA-HRP mouse antibody (1:1000 dilution). TbAKD299V strain was used as positive control since it harbours a AK-HA tagged protein of about 37kDa, Nysm was used as negative control. TbAMPKβ was tested at several concentrations of crude protein extract from 1,2 and 4 millions of cells respectively. Nysm: New York single marker cell line. D299V: Nysm carryingHA-tagged Adenosine kinase. Nysm-β-SSH (clone 1): Nysm with TbAMPKβ subunit tagged in C-terminus with StrepII-StrepII-HA tag.

Replacement of endogenous TbAMPKγ by StrepII-StrepII-FLAG tagged variant (Nysm-γ-SSF)

In parallel to the TbAMPKβ-SSF construction, the construct needed for replacing endogenous TbAMPKγ with a SSF tagged variant harbouring the hygromycin resistance marker was created. The final 200 bp of TbAMPKγ gene without the STOP codon and the 200 bp of the beginning of 3’UTR of TbAMPKγ gene were amplified with the necessary restriction enzyme sequences. After digestion and ligation into the pUC57_Nysm-TbAMPKβ-SSF plasmid the resistance cassette (puromycine) was replaced by to hygromycine (pUC57_ Nysm-TbAMPKγ-SSF). The plasmid was achieved, but due to observation of false positive recognition of proteins in parasite extracts using anti-FLAG antibody and absence of StrepII antibody detection, we refrained from further work on this construct and decided to change the tag.

Replacement of endogenous TbAMPKγ by cMyc tagged variant (Nysm-γ-cMyc)

The final tag chosen for the TbAMPKγ subunit was the cMyc tag. After failing to anneal two primers containing the triple cMyc label and ligate it into the final plasmid, the label was amplified from pMOTag43M [7] which contains the triple tag of interest. The sequence was amplified and inserted into pUC57_Nysm-TbAMPKγ-SSF providing plasmid pUC57_Nysm-TbAMPKγ-cMyc.

The final construct digested by XbaI and BamHI, was ethanol-purified and successfully electroporated into Nysm cell line as described previously. Five clones (A-E) resistant to hygromycine antibiotic were tested for correct insertion of the tagged protein gene sequence by PCR between the TbAMPKγ gene and the α-β tubulin intergenic region.

(Figure 31).

Figure 31 Validation of AMPK-cMyc tag insertion for clones A-E via PCR from gDNA and Actin control. Nysm: New York single marker cell line. Nysm-γ-cMyc (clone A-E): Nysm with TbAMPKγ subunit tagged in C-terminus with cMyc tag.

To verify the expression of the tagged protein, after extraction of the total crude protein from all clones, the protein was tested with the anti-cMyc antibody. A HEK cell extract, containing a 50 kDa protein tagged with cMyc, was used as a positive control. Western blot analysis shows that all clones express the tagged protein around 60 kDa corresponding to the expected size, but at different intensities (Figure 32). For further immunoprecipitation analysis (2.3.2) clones A and B were selected.

Figure 32 Validation of TbAMPKγ-cMyc expression in clones A-E. Blots were probed against cMyc epitope (A) and against the loading control TbBip (B). Nysm: New York single marker cell line. Positive control: Total protein extraction from HEK cells harbouring a 50kDa cMyc tagged protein. Nysm-γ-cMyc (clone A-E): Nysm with TbAMPKγ subunit tagged in C-terminus with cMyc tag.

Double mutant strain harbouring TbAMPKβ-SSH and TbAMPKγ-cMyc

Simultaneously to the preparation of the strain containing the TbAMPKγ subunit tagged with a triple cMyc, the production of a double-tagged strain expressing simultaneously TbAMPKβ-SSH and TbAMPKγ-cMyc was achieved. The cell line harbouring the TbAMPKγ-cMyc tagged protein described above was electroporated with the XbaI/BamHI-digested fragment of the construct containing the TbAMPKβ-SSH sequence. Two clones were resistant to both puromycine and hygromycine antibiotics.

The correct insertion of the tag was verified by PCR from gDNA as previously

The correct insertion of the tag was verified by PCR from gDNA as previously