2.2 METHODS
2.2.2 Biochemical methods
2.2.2.1 Protein expression and purification
Glutathione-S-transferase (GST) fusion proteins were expressed in E.coli transformed with expression vectors pGEX-4T harboring the gene of interest. O/N bacterial cultures were diluted 1:40 in 1 liter LB broth containing selective antibiotics and incubated at 37 °C shaking until OD600 % 0,7. Protein expression was induced by addition of 1 mM
isopropyl-"-D-thiogalactopyranoside (IPTG). Culture was incubated for 3 hrs at 37 °C with shaking and then centrifuged (6000 rpm, 4 °C, 15 min). The pellet was resuspended in 10 ml cold lysis buffer and the suspension was sonicated on ice 10 times for 10 sec with 17 % amplitude. The lysate was centrifuged (20.000 g, 4 °C, 15 min), the supernatant mixed with washed 1 ml glutathione sepharose 4B beads and incubated for 1 h at 4 °C rotating end over end. Using a column, unbound proteins were removed by addition of cold washing buffer. GST-fused proteins were eluted by adding 0,5 ml cold elution buffer.
When necessary, protein samples were dialyzed against 50 mM Tris-HCl (pH 7,4), 10 mM MgCl2 at 4 °C O/N.
2.2.2.2 Determination of protein concentration
Total protein concentration in samples was measured using the Bradford method based Protein Assay (Bio-Rad) according to the manufacturer’s protocol. This colorimetric protein assay is based on an absorbance shift due to protein binding of the dye Coomassie Brilliant Blue G-250. Absorbance was measured at 595 nm.
2.2.2.3 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Separation of proteins according to their molecular weight was carried out with denaturing SDS-PAGE. Electrophoresis was performed with mini gels (6 x 9 cm) in a Mini Protean II apparatus. Preparation of resolving and stacking gels is described in Table 2.6.
Protein samples for analysis were mixed with 3x SDS-sample buffer and heat-denatured for 10 min at 95 °C. Gels were run at a constant voltage of 120 V in 1x SDS-running buffer. Prestained protein ladder was used for protein weight standard. After electrophoresis proteins were either visualized using Coomassie staining (2.2.2.4) or transferred onto polyvinylidene difluoride (PVDF) membranes (2.2.2.5).
Table 2.6: Preparation of polyacrylam ide gels.
Resolving, 10 ml Stacking, 3 ml
10 % 12 % 5 %
ddH20 4 ml 3,3 ml 2,1 ml
Acrylamide (30%) 3,3 ml 4,0 ml 0,5 ml
Resolving buffer 2,6 ml 2,6 ml -
Stacking buffer - - 0,41 ml
10% Ammonium persulfate (APS) 0,1 ml 0,1 ml 0,03 ml
Tetramethylethylenediamine (TEMED)
0,004 ml 0,004 ml 0,003 ml
2.2.2.4 Coomassie staining
To visualize proteins after electrophoresis (2.2.2.3), the gel was rinsed in ddH20 before being soaked in Coomassie staining solution for 3-16 hrs. After staining, the gel was destained by periodic exchange of spent destaining solution with either destaining solution or ddH20 until protein bands became clearly visible.
2.2.2.5 Western blot
For specific analysis of proteins by immunodetection proteins were transferred onto a PVDF membrane after electrophoresis (2.2.2.3). PVDF membrane was activated in methanol before being soaked in transfer buffer. Six Whatman filter papers were also soaked in transfer buffer. The blot was assembled from cathode to anode: 3 Whatman
filter papers, gel, membrane, and 3 Whatman filter papers. Transfer was carried out in a semi-dry blotting apparatus at constant current of 2 mA/cm2 for 1 h.
2.2.2.6 Immunodetection
To detect proteins that were immobilized on PVDF membranes with specific antibodies the membrane was initially incubated in blocking buffer for 1 h at room temperature (RT).
Binding of the primary antibody was carried out in blocking buffer (Table 2.4) at 4 °C overnight under gentle agitation. After 3 washing steps for 5-10 min with PBST or TBST the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Table 2.5) for 1 h at RT under gentle agitation. After 3 additional washing steps for 5-10 min each with PBST or TBST detection was performed using chemiluminescence-based SuperSignal West Femto substrate according to the manufacturer’s protocol. For documentation, light sensitive X-ray films were used.
2.2.2.7 Enzyme-linked immunosorbent assay (ELISA)
To detect production of human interleukin 8 (IL-8) by CNF1-intoxicated HeLa cells, ELISA was performed in accordance with the manufacturer’s instructions (BD OptEIA, BD Bioscience). Briefly, cells were intoxicated with CNF1 (300 ng/ml) for indicated time points at 37 °C. Culture medium was collected and centrifuged (10.000 g, 5 min, 4 °C).
Supernatants were added to a 96-well plate coated with anti-human IL-8 antibody. For detection of captured IL-8, biotinylated detection antibody and streptavidin-conjugated horseradish peroxidase conjugate were used. Captured IL-8 was visualized using a tetramethylbenzidine (TMB) substrate reagent set and absorbance was read at 450 nm in a microplate reader. Recombinant human IL-8 was used as a standard. IL-8 levels were determined by interpolation from the standard curve.
2.2.2.8 Whole cell lysates
Cells were washed 2x with ice-cold PBS (+ Ca2+/Mg2+) and scraped off in cold cell lysis buffer. Cell lysates were centrifuged (20.000 g, 20 min, 4 °C) and supernatants were collected for analysis.
2.2.2.9 Rho GTPase activation pulldown
Activation of Rho GTPases can be analyzed and quantified in cell lysates with pulldown assays. Specific effector proteins of the active form of the Rho GTPase are used as GST-fusion proteins. The GST-GST-fusion proteins were recombinantly expressed and purified from E. coli (2.2.2.1). To analyze active Rac1, GST-PAK-CRIB was used whereas GST-ELMO-2NT was used to pull down active RhoG. Glutathione sepharose 4B beads (100 µl) were washed 3 times by centrifugation (510 g, 3 min, 4 °C) with cold PBS containing 100 µM phenylmethylsulfonylfluorid (PMSF) followed by incubation with GST-fusion proteins for 1 h at 4 °C rotating. Beads coupled with GST-fusion proteins were washed three times with cold PBS/PMSF and incubated with cell lysates (2.2.2.8) for 2 hrs at 4 °C rotating. After 4 washing steps with cold PBS/PMSF, remaining buffer was completely removed from beads and proteins were released with 3x SDS-sample buffer. Beads were incubated with 30 µl
3x SDS-sample buffer for 10 min at 95 °C and the supernatant was transferred into a new tube.
2.2.2.10 Nucleotide binding assay
Fluorescent nucleotide analogs incorporating N-methylanthraniloyl (mant) fluorophore were used to analyze functional GDP-binding ability of recombinant Rho GTPases. The fluorescence intensity of GTPase-bound mant-GDP is higher compared to free mant-GDP and therefore nucleotide-protein interactions are detectable. The fluorescence of baseline mant-GDP (0,5 µM final concentration) in CNF1 buffer was monitored with excitation at 350 nm and emission at 448 nm. Measurements were taken every 30 sec. After the addition of 1 µM Rho GTPase, the increase in fluorescence intensity was recorded. Relative fluorescence intensity was calculated as the fluorescence signal of GTPase-bound mant-GDP divided by fluorescence signal of baseline mant-mant-GDP.
2.2.2.11
In vitro modification of recombinant Rho GTPases by CNF1For analysis of Rho GTPase deamidation, recombinant proteins of GST-CNF1 and GST-Rho GTPase (RhoG, RhoA) were incubated for 4 hrs at 37 °C in CNF1 buffer. The ratio of CNF1:GTPase (molar masses) varied between 5-20:1. After incubation, samples were supplied with 3x SDS-sample buffer and proceeded for SDS-PAGE.
2.2.2.12 Proteolytic digestion for mass spectrometric analysis
The gel was stained with colloidal Coomassie, the bands were cut out, the proteins were reduced with 10 mM DTT at 56 °C for 30 min, the cysteine residues were modified with 55 mM iodacetamid for 20 min in the dark and the proteins were in-gel digested with 5 ng/µl trypsin in 50 mM NH4HCO3 at 37 °C for 16 hrs. After digestion, the gel pieces were repeatedly extracted (50 % acetonitrile/5 % formic acid), the combined extracts dried down in a vacuum concentrator and redissolved in 20 µl formic acid (0,1 %).
2.2.2.13 Mass spectrometry
Liquid chromatography/mass spectrometry (LC/MS) runs were done on a Quadropol-Tof-Tandem mass spectrometer equipped with Ultra Performance LC® (Waters, Eschborn, G).
Samples were applied onto a trapping column, washed for 10 min (flow: 5 µl/min) with 5
% acetonitrile, 0,1 % formic acid and then eluted onto the separation column with following gradient: A = 0,1 % formic acid and B = 0,1 % formic acid in acetonitrile, 5 – 50 % B in 120 min.
In order to identify and label-free quantify the proteins, the MSE technique according to Silva et al. (2005) was applied: alternating scans (0,95 sec; 0,05 sec interscan delay) with low (4 eV) and high (ramp from 20-35 eV) collision energies were recorded. The data were evaluated with the software package Protein Lynx Global Server 2.3 (Waters, Eschborn, G).