BIOCHEMICAL METHODS

3.1 Protein expression and purification

Plasmids were transformed into E. coli BL21 using a heat shock procedure. For protein expression, over night bacterial cultures were diluted 1:20 in Luria Bertani medium (LB) containing the selective antibiotic and grown at 37 °C to an OD600 of 0.6 to 0.8. Bacterial

cultures were shifted to 18 °C and protein expression was induced by 0.4 mM IPTG (isopropyl- b-D-thiogalactopyranoside) for 3 to 4 hours. Cells were harvested (3000 g for 10 min at 4 °C) and french-pressed or sonificated in lysis buffer containing 50mM Tris, pH7.4, 100 mM NaCl, 10% glycerol (and additional 0.1% Triton X-100 for purification of GST- YopOwt and GST-YopOK269A).

The soluble fractions with the GST fusion protein from bacterial lysates were rapidly purified with Glutathione-Sepharose 4B (Amersham, Freiburg, Germany). The principle is based on the strong affinity of the GST protein for Glutathione, allowing other proteins to flow through the column packed with the Glutathione-Sepharose beads. Through several wash steps with lysis buffer, the unspecific bound proteins are washed through the column. The GST-fusion protein was either eluted under mild conditions with lysis buffer containing reduced glutathione (30mM glutathione pH8.8 in lysis buffer) or cleaved overnight at 4 °C with 50 units of thrombin per ml settled beads.

If required (e.g. for microinjection) supernatants containing the desired protein were dialyzed against buffer containing 10 mM Tris pH 7,5, 150 mM NaCl, 2 mM MgCl2, 0.1 mM DTT.

3.2 GST-Invasin expression for coating of fluorescent beads

For bacterial expression of invasin (Schulte, Grassl et al. 2000; Schulte, Kerneis et al. 2000; Wiedemann, Linder et al. 2001) E. coli BL21 harboring pGEX-4T-3 with an insertion of the

inv397 gene (BL21-inv397), coding for the 397 carboxy-terminal amino acids of invasin, was grown at 24 °C in LB to an OD of 0.7. Expression of the GST-Invasin fusion protein was induced with IPTG (isopropyl- b-D-thiogalactopyranoside) at a final concentration of 0.3 mM. Cells were grown for 3 additional hours at 37 °C before being harvested by centrifugation and frozen at -20 °C. Frozen cells were resuspended in lysis buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 10 % glycerol) supplemented with complete protease inhibitor mix (Roche, Mannheim, Germany) and disrupted by French press. Lysates were cleared by centrifugation and GST-Inv397 was purified with glutathione Sepharose 4B (Amersham, Freiburg, Germany). GST-fused protein was eluted with reduced glutathione (30 mM, pH 8.9, in lysis buffer).

3.3 Determination of protein concentration (Bradford)

Concentration of solubilized protein was determined with the BioRad protein Assay (BioRad, Munich, Germany) based on the method of Bradford. The Bio-Rad Protein Assay is a dye- binding assay in which a differential color change of a dye occurs in response to various protein concentrations. The absorbtion maximum for the used acidic solution of Coomassie Brilliant Blue G-250 dye shifts from 465 nm to 595 nm upon binding to protein. As a standard bovine serum albumin (BSA, Sigma-Aldrich, Munich, Germany) was used in the concentrations 20 µg/ml, 15 µg/ml, 10 µg/ml, 5 µg/ml und 1 µg/ml. To 800 µl of sample or standard solution 200 µl of dye reagent were added and after vortexing and an incubation of 5 minutes at room temperature the absorbance at 595nm was measured.

3.4 SDS/PAGE

In SDS/PAGE (Sodium-dodecyl-sulphate/polyacrylamide gel electrophoresis) proteins are separated on the basis of their molecular weights as they migrate through a gel. SDS is an anionic detergent that denatures proteins. The SDS also disrupts hydrogen bonds, blocks hydrophobic interactions, and substantially unfolds the protein molecules by eliminating the tertiary and secondary structures. In the discontinuous gel system employed in this work, a stacking gel (non-restrictive large-pore gel, 5% acrylamide) was layered on top of a separating gel (resolving gel, 8-12% acrylamide). Samples were mixed with 5x Laemmli SDS loading buffer (2-mercapto-ethanol added for a final concentration of 5 %) incubated at 95 °C (5 min) and subjected to electrophoresis. With the aid of a protein marker (prestained high range maker, Peqlab, Erlangen, Germany) the molecular weight of the proteins applied could be estimated.

5x SDS-sample buffer (Laemmli)

0.5 M Tris/HCl pH 6,8 5 ml

Glycerol 2 ml

SDS 0.8 g

0.5 % Bromophenol blue 1 ml

Stacking Gel (5%)

ddH2O 5.5 ml

0.5M Tris-HCl, pH6.8 1 ml

30% acrylamide 1.3 ml

10% SDS 800 µl

10% ammonium persulfate (APS) 800 µl

TEMED (Sigma) 8 µl Resolving Gel (10%) ddH2O 7.9 ml 0.5M Tris-HCl. pH8.8 5 ml 30% acrylamide 6.7 ml 10% SDS 200 µl

10% ammonium persulfate (APS) 200 µl

TEMED (Sigma) 8µl

SDS/PAGE running buffer (10%)

Tris-Base 30.3 g

Glycine 144 g

SDS 10 g ad 1 l ddH2O

3.5 Coomassie Staining

Gels were stained for 30 minutes in Coomasie staining solution (0.1% Coomassie Brilliant blue , 25% methanol, 10% acetic acid) and subsequently destained in Destain solution (33% [v/v] Methanol, 10% [v/v] glacial acetic acid).

3.6 Western Blot

Western blot is a technique that employs electrophoretic transfer of proteins separated by SDS/PAGE to a membrane and subsequent detection of the proteins by antibody-coupled chemiluminescence or autoradiography. The separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Immobilon–P, Millipore, Schwalbach, Germany) by electrophoretic transfer. For this, a SDS/PAGE gel was placed directly on a piece of methanol-activated PVDF membrane (10 seconds in 100% methanol) and "sandwiched" between two electrodes. For tank-blotting the gel and the membrane were submerged in a container (Mini Protean-II, BioRad, Munich Germany) with blotting buffer (25 mM Tris- Base, 96 mM Glycin) and an electric field applied (100V, 100min or 30V over night, 4 °C).

For semi-dry-blotting the gel and the membrane were transferred to a semi-dry blotting device and proteins transferred onto PVDF membrane for 1 hour at 1.2 mA/cm2.

The PVDF membrane was blocked for 1 hour or overnight at room temperature or 4 °C, respectively, in PBS pH 7.4, 3% bovine serum albumin (BSA), 0.05% Tween 20. The membrane was incubated with primary antibody for 1 hour at room temperature (or 4 °C overnight), washed three times for 5 minutes followed by incubation with horseradish peroxidase-coupled secondary antibody (1:2000 to 1:10.000) for 1 hour. After a final washing step the detection solution (ECL Western blotting detection reagent, Amersham Biosciences, Freiburg, Germany) was applied according to the protocol of the manufacturer. Detection is based on a chemoluminescence reaction in which Luminol is oxidized in the presence of H2O2 by the peroxidase conjugated to the secondary antibody. The emitted light was detected by exposing the membrane to an X-ray film (Kodak, Stuttgart, Germany). The exposed film was developed in a processor for X-ray film development (Agfa Curix 60) and, in case of quantification of chemiluminescence signals, two dimensional densitometry was performed using a Gel Doc EQ System with Quantity One software (BioRad, Munich, Germany).

3.7 Kinase Assay and Kinase Quantification

Kinase reactions were performed with 3-10 µg of GST-YopO protein or indicated fragments in 20 µl kinase buffer containing 20mM Hepes pH 7.4, 1 mM ATP, 1mM DTT, 10 mM MgCl2, 2 mM MnCl2 supplemented with 5µCi [γ-32P] adenosine 5’ trisphosphate (Amersham Biosciences, Freiburg, Germany) and 3-10µg G-actin from Dictyostelium discoideum (generous gift from Michael Schleicher).

Reactions were incubated at 30 °C for 30 minutes and phosphorylation was stopped by addition of SDS/PAGE sample buffer and heating for 5 minutes at 95 °C. Reactions were subjected to SDS/PAGE and/or Western blot transfer and radioactivity in dried gels or on membranes was visualized and quantified using a Phosphoimager (FLA 3000, Fujifilm, Tokyo, Japan) with Aida 4.0 Image Analyzer software (Raytest, Straubenhardt, Germany) or X-ray film (Kodak, Stuttgart, Germany). For autoradiography quantification of single and double point mutant YopO proteins autophosphorylation of YopO89-729 was set to 100% and autophosphorylation of mutant proteins normalized.

3.8 Phosphorylation of artificial substrates

Kinase reactions were performed as described (see above) with designated artificial substrates (histone and myelin basic protein, MBP) added to kinase assay, run on SDS/PAGE and subjected to autoradiography.

Reactions were incubated at 30 °C for 30 minutes and phosphorylation was stopped by addition of SDS/PAGE sample buffer and heating for 5 minutes at 95 °C. Reactions were subjected to SDS/PAGE and/or Western blot transfer and radioactivity in dried gels or on membranes was visualized and quantified using a Phosphoimager (FLA 3000, Fujifilm, Tokyo, Japan) with Aida 4.0 Image Analyzer software (Raytest, Straubenhardt, Germany) or X-ray film (Kodak, Stuttgart, Germany).