2.4.1 Protein sample preparation
2.4.1.1 Bacterial cultures
0.5 ml of bacterial cultures were pelleted at 12000xg for 2 mins per condition, and supernatant removed. As a general rule, if analysing protein expression, one sample was taken for the uninduced control and two samples for the IPTG
induced (see below). The complete uninduced sample and one of the induced were resuspended in 60 l of 1x protein loading buffer (250 μl 4x NuPAGE® LDS sample buffer [Invitrogen], 750 μl 1x PBS and 25 μl β-mercaptoethanol), 10
seconds of sonication were applied, and samples were denatured at 95 oC for 10
mins. Samples could be stored at -20 oC until being used. For the other induced sample, it was resuspended in 45 l of PBS, subjected to 3 cycles of sonication (10 seconds each) in order to obtain the soluble fraction and centrifuged again 12000xg for 10 mins. Supernatant (soluble fraction) was transferred to a
different tube and supplemented with 15 l of 4x NuPAGE® LDS sample buffer (Invitrogen). Pellet (insoluble fraction) was supplemented with 60 l of 1x protein loading buffer.
2.4.1.2 Trypanosome cultures
As a general rule, between 106-2.5x106 parasites were used per sample. They were centrifuged at 1500 x g for 10 mins and lysed with 20 l 1x protein loading buffer per sample before being denatured at 95 oC. Samples could be kept at -20
oC until needed.
2.4.2 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Sodium dodecyl sulphate polyacrylamide gel electrophoresis was used to
separate proteins according to size. NuPAGE™ Novex™ 10% Bis-Tris Protein Gels, 1.0 mm, 12-well (Invitrogen) were generally used in a XCell SureLock™ Mini-Cell Electrophoresis System (Invitrogen). NuPAGE® MES SDS Running Buffer or
NuPAGE® MOPS SDS Running Buffer (Invitrogen) were used as running buffer. Samples were defrosted and denatured again at 95 oC before loading 20 l per
lane. Electrophoresis was run for 45 mins at 150 V.
To visualize proteins after electrophoresis, polyacrylamide gels were rinsed in milliQ water and SimplyBlue™ SafeStain ready-to-use reagent (Life technologies) was applied for about one h followed by three 20-minute rinses with distillate water. To fasten the process, the reaction was warmed up for 10 seconds in the microwave at maximum power.
2.4.3 Western blotting
For western blot, proteins separated by SDS-PAGE were transferred to a Polyvinylidene difluoride (PVDF) Biotrace membrane (Pall) in an X-Cell II Blot
Module (Invitrogen). PVDF membrane was activated in methanol for 30 seconds, rinsed in milliQ water then equilibrated in transfer buffer [20 % (v/v) methanol, 20 mM Tris, 15 mM glycine]. The transfer chamber was assembled and filled with transfer buffer with all pads and filter papers soaked previously in it. Transfer was performed at 30 V for 2 h with the outer chamber filled with water to
provide a coolant. As a general rule, membrane was rinsed in 1x PBST (PBS, 0.01 % Tween-20 [Sigma]) for 10 mins on a rocker then incubated for 1 h in blocking solution (1x PBST, 5 % Milk powder [Marvel]) or if required, overnight at 4 oC. After, the membrane was rinsed once for 10 mins in 1x PBST then it was moved to blocking buffer containing the required primary antisera and incubated for 1 h. Then, membrane was washed three times for 10 mins with 1xPBST, and incubated for another hour with a secondary antibody against the primary
serotype conjugated to horseradish peroxidase (HRP). Again, the membrane was washed three times for 10 mins with 1 x PBST then the SuperSignal West Pico Chemiluminescent Substrate (Thermo-Fisher) or ECL Prime Western Blotting Detection Reagent (Amersham) were pipetted evenly onto the membrane surface, which was subsequently incubated in the dark for 5 mins. The
membrane was then exposed in a gel doc, or using medical X-ray blue film MXB (Carestream) in a Kiran cassette for ~one second to overnight and films were developed using a Kodak M-25-M X-omat processor.
2.4.3.1 Antibody detection
Table 2-8. List of antibodies used for western blot in this thesis.
Antibody Serotype Clone Concentration Supplier
VSG 221 variant Rabbit IgG N/A 1:20000 R. McCulloch
VSG crossreactive* Rabbit IgG N/A 1:20000 D. Horn
EF1 Mouse IgG CBP-KK1 1:25000 Millipore
EP procyclin Mouse IgG TBRP1/247 1:500 Cedarlane
Myc Mouse IgG 4A6 1:7000 Millipore
HA Mouse IgG HA-7 1:1000 Sigma
Mouse IgG Goat IgG N/A 1:5000 Novex
Rabbit IgG Goat IgG N/A 1:5000 Novex
*Required hypotonic lysis to maintain folding of the crossreactive determinant (10 mM Tris base, pH 7.5 with HCl on ice) and centrifugation 12000xg 2 mins to separate supernatant separating soluble and insoluble fractions.
^The HA antibody was incubated overnight at 4 oC
Abbreviations: VSG (Variant Surface Glycoprotein), EF1(Elongation Factor 1, Ig (Immunoglobulin).
2.4.4 Protein expression and purification
All proteins expressed for this thesis were mutants derived from the same Trx- RDK2 protein and were expressed under the same conditions. Codon-enriched
Escherichia coli RosettaTM (DE3) p-lysS strain, with T7 lysozyme to supress basal
T7 expression, was used as the bacterial host. Protein expression was induced with 1 mM IPTG in 1L log-phase cells (OD600=0.5) at 17oC overnight.
Proteins were derived from pET32 Xa/LIC expression system (Novagen). They all express the protein of interested fused to a N-terminal thioredoxin tag for solubility and a 6xHis tag for purification using ion metal affinity
chromatography (IMAC).
After centrifugation of an overnight induced culture, 14000g for 15 mins pellets were lysed with B-PER (Thermo Scientific). Then they were sonicated (5 cycles of 10 seconds on ice), centrifuged again to collect supernatant, and filtered through a 0.22 m minisart (Sigma), and loaded for IMAC in a 5 ml HisTrapTM HP
column (GE Healthcare), precharged with Ni2 SepharoseTM High Performance medium on an AKTA purifier (GE Healthcare). All buffers used in the AKTA system were previously filtered through a 0.22 m Stericup® filtration unit (Millipore). The column was equilibrated with 20 mM NaH2PO4, 500 mM NaCl, 5
mM imidazole, pH 7.4. After binding the 6xHis-tag, the column was washed with the same buffer but containing 35 mM imidazole. Finally, elution was conducted with the same buffer but including 500 mM imidazole monitoring UV absorbance in 1 ml fractions to detect the protein content. Spaced fractions across the elution peak were sampled and run in a 12% SDS-PAGE and stained with
SimplyBlue™ SafeStain ready-to-use reagent (Life technologies) to assess purity of Trx-RDK2 (and derivatives) in order to pool the best fractions. Imidazole was removed from the pooled fractions in a PD-10 desalting column. To purify the protein further, the sample was loaded into a pre-equilibrated anion exchange HiTrap Q XL column in the AKTA purifier, washed with 10 column volumes of 25 mM Tris-HCl pH 7.9 and eluted in the same buffer with a linear gradient 0-1M NaCl over 10 column volumes. Fast Protein Liquid Chromatography (FLPC) was monitored with UV absorbance, in order to sample fractions matching the
elution peak for SDS-PAGE and SimplyBlue™ SafeStain ready-to-use reagent (Life technologies). Proteins were collected in 6-8 ml of buffer and were
concentrated with 30 KDa molecular weight cut-off Vivaspin Turbo 15 columns (Sartorius).
Protein concentration was assessed both in Nanodrop 1000 (Thermo Scientific) and via Bradford assay (Bradford, 1976). Nanodrop was chosen for systematic measurements as it was more straightforward and provided the same range of concentrations.
All proteins were stored by immersion in liquid nitrogen and storage at -80oC with 10% glycerol.
2.4.5 Protein kinase assay
Purified, recombinant Trx-RDK2 and derivative proteins (active, dead mutants and phosphosite mutations) were adjusted to 100 g/ml in 20 l of kinase assay buffer (KAB) (50 mM MOPS, 20 mM MgCl2, 10 mM DTT, 10 mM EGTA), i.e. 2g of
protein per reaction tube. 2 reactions were prepared per protein to be assayed with and without a substrate. 20 l of kinase assay mastermix (KAM) were added to 100 M ATP, 0.37 MBq [-32P]-ATP, and 10 mg/ml of -casein (Sigma-Aldrich) as a substrate if applicable in KAB. Final assay concentration of the protein was 50 g/ml. Reactions were incubated at 30oC for 30 mins with shaking and
stopped by the addition of 40 l 2x SDS-PAGE buffer and boiling for 3 mins. 20 l of this was separated by SDS-PAGE, the gel was dried and placed into a cassette with a phosphor-storage screen (Molecular Dynamics) for 8 to 72 h before
visualising the screen in a Typhoon 9400 imager (GE Healthcare). Alternatively, it was exposed for 72 h at -80 oC with a medical X-ray blue film MXB
(Carestream) in a cassette with a calcium tungstate-intensifying screen (Kiran). Films were developed in a Kodak X-Omat processor.
2.4.6 Xa factor cleavage
In order to choose the right conditions, before scaling up, Xa factor (Novagen) cleavage properties were assayed in a miniaturized screen testing 0, 0.1, 0.2, and 0.5 U/l against 10 g of Trx-RDK2. Incubation times were optimized also testing 2, 4, 8, and 16 h in 50 l reactions. 10 l aliquots were mixed with 10 l 2x loading buffer, boiled for 5 mins, separated SDS-PAGE and stained with SimplyBlue™ SafeStain ready-to-use reagent (Life technologies). After analysing results, 6 h at 0.5 U/l was used at a large scale to cleave Trx-RDK2 and Trx- RDK2K70M.