Biochimie II. Assistants Ben Brankatschk Eleonora Torti

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1 Biochimie II Purification de protéines exprimées dans des cellules humaines en culture Daniel Abegg Christophe Berthier Pauline Bonvin Assistants Ben Brankatschk Eleonora Torti Université de Genève, Science II, Laboratoire 104A Groupe A December 1, 2008 Introduction In this experiment we are interested in the proteins which can interact with Rab5 (EEA1) and Snx12 (Snx3 and YIPF5). Rab5 belongs to the monomeric GTPase superfamilly (Rab) and is regulated by GEF (guanine nucleotide exchange factors) from its inactive form binding GDP to an active form with GTP[6]. Rab5 plays an important part in membrane traffics by mediating early endosomes in combination with SNAREs proteins to their target[5]. One factor which interact with Rab5 is EEA1 (early endosome antigen 1) and is an effector of Rab5 required for the endosome fusion[1]. Snx12 also plays a role in the membrane traffics but its exact function is unknown. It only contains a PX domain (phosphoinositol-binding domain) which binds the target protein to membranes like early endosomes.[2]. Snx3 and YIPF5 are known to interact with Snx12. Snx3 has a very similar primary structure with Snx12. It also only has the PX domain[4] and we think that it s function is probably close to that of Snx12. YIPF5 functions as an adaptor in traffic regulation[3] between the endoplasmic reticulum and Golgi[7]. Cells were transfected with tags, GFP for Rab5 and myc for Snx12. Those two proteins were than purified through immunoprecipitation with antibody against the tag and A proteins on sepharose beads which precipitate GFP-Rab5 and myc- Snx12. Rab5 and Snx12 were released from the antibody with β-mercaptoethanol. An electrophoresis and a Western blot were done to separate them form each other to be able to do an immunodetection. The nitrocellulose membrane (which was cut in tree parts) was incubated with primary antibodies against EEA1, GFP and myc- Snx12 and than with a secondary antibody coupled to HRP. Lastly we analyze the results with ECL. 1

2 Material and Methods In this experiment, we manipulate some epithelial cells called HeLa. This kind of cells must have grown in adapted medium. It means that the medium has to be sterile. The principle is that the cells cover up the total surface of the dish. Then a protease called trypsin is used to take off the cells from the surface and to separate them one from the other to finally transfer them in another container. This last procedure is called passage. To obtain a good passage, the medium of the cells has be aspirated then washed two times with PBS at 37 C. Afterwards, 5 ml of trypsin are added into the dish for 30 sec. 4 ml of trypsin are taken out of the medium. 4 ml of the cell s medium are added and diluted by four. 24 hours after the passage, the transfection is possible. For the transfection, a tube containing 300 microlitres of OptiMEM +Fugene and another one with 3 µg of DNA are mixed up and added drop by drop into the cells. The following step is the cell lysis and the immunoprecipitation. The dishes are washed three times with PBS. Then 1 ml of lysis buffer is added and the cells are detached by scratching the surface of the dish. The containing of the dishes are transferred in two tubes and stirred during a few minutes. Then the tubes are centrifuged and the supernatant of tubes is recovered. After that an aliquot of 40 microliters is taken from supernatant. The rest is put with Sepharose-protein A beads, centrifuged and stirred. Then the supernatant is separate in two equal volumes and one is incubated with antibodies during 1 hour à 4 C than and 100 microliters of Sepharose-protein A beads are added to the two supernatant. The containing is as well stirred and centrifuged. The supernatants are wash 3 times with lysis buffer (without NP40) and centrifuged each time in-between. 25 µl of charge buffer (contains β-mercaptoethanol) is added and the supernatants are heated to 100 C during 5 min. The next step is to analyze the proteins purified by SDS Polyacrylamide Gel Electrophoresis. At the time the gel is prepared, it is immerged in the running buffer. The samples containing the proteins and markers are dropped in the right migration sites. The migration takes around 1h30 at 100 V. The next step is the transfer of the proteins on the membrane PVDF. Papers Wathman make a sandwich in which the membrane and the gel remains. An electric field is applied on the sandwich to have the proteins migrate on the PVDF membrane. The last step of this experiment is the analyze of the blots. First, the membrane is incubated in a saturation buffer (TBS- Tween with 5% milk) which allows the membrane to not fix any antibodies on unspecific sites. Afterwards, the membrane is incubated for an hour with an antibody which detects the GFP marker on the analyzed proteins. The membrane is washed with TBS-Tween, incubated again with another antibody called anti-igg and diluted 1/3000 in TBS 5% milk. Finally the membrane is incubated one more time with ECL. This procedure allows by chemiluminescence to show the positions of the proteins. We want to detect here Rab5 and Snx12. Results After the SDS-PAGE and the Western Blot, we obtained the results showed in figure 1. The antibody used for the immunoprecipi- 2

3 Figure 1: Western Blot; left part: cells transfected with the GFP-Rab5 plasmid; right part: cells transfected with the myc-snx12 plasmid tation is written on the top of the gel and the one used for the Western blot on the sides of the gel. The input lanes were loaded with the entire cell lysate, meaning that they contain all the proteins present in the cells. The standards of molecular weight are illustrated in the figure 2. just below 50 kda and the other at about 30 kda. The same bands are visible in the input lane in which there are also three less dark bands at 25, 40 and 180 kda. In the without antibody lane, only one band is present, at the same molecular that the lightest band in the antibody lane. For the myc-snx12 protein, the same band is visible in the three lanes (input, antibody, no antibody) at approximately 26 kda. Disscusion GFP-Rab5 Protein Figure 2: Standard of molecular weight used in the SDS-PAGE The lysate of the Rab5 transfected cells was immunoprecipitated with an anti-gfp antibody and, after running the gel, cut in two parts which were treated respectively with an anti-eea1 and an anti-gfp. The molecular weights of the proteins are summarized in the table 1. For the GFP-Rab5 protein, two large bands can be seen in the antibody lane, one 3

4 Protein Molecular Weight [kda] Rab GFP 27 GFP-Rab EEA1 162 Table 1: Molecular weight of the proteins implicated in the GFP-Rab5 Western Blot We can now identify the bands seen on the gel: Lane 1: the band at 50 kda must be the GFP-Rab5 protein and the one at 30 kda the GFP alone. It is very probable that the GFP-Rab5 protein was cleaved during the experiment and then migrated alone in the gel. The band at 180 kda is most likely the EEA1 present in the cells. The two other light bands are certainly due to unspecific interactions between some proteins in the cells and the α-gfp antibody. The antibody should possibly binds in a less precise way with other domains of proteins that the GFP one and this unspecific protein are visible on the gel. Lane 2: as explained for the first lane, the 50 kda band is the GFP-Rab5 protein and the 30 kda band the GFP cleaved off. Lane 3: we didn t put any antibody in the lysate which was loaded into this lane, so nothing is supposed to bind to the beads. For this reason, we weren t awaiting any band in this lane but we can see a small band at 50 kda which seems also to be the GFP-Rab5 protein. We can suppose that a domain present either on Rab5 or on GFP is able to interact unspecificly with the beads (with the A protein or with the sepharose). As the interaction is not perfect, only few proteins bind to the beads and the obtained band is very light. Myc-Snx12 Protein The lysate of the Snx12 transfected cells was immunoprecipitated with an anti-myc antibody and, after running the gel, treated again with the anti-myc (figure 1). The molecular weights of the proteins are summarized in the table 2. Protein Molecular Weight [kda] Snx myc tag ca. 2 myc-snx Table 2: Molecular weight of the proteins implicated in the myc-snx12 Western Blot We can now deduce that the 26 kda band present in the gel must be the myc-snx12 protein. We have it in the lanes 4 and 5, like awaited, but also in the lane 6. This lane should theoretically be empty because the antibodies weren t added and nothing was supposed to bind the beads. But it seems that the myc-snx12 protein can bind directly the beads and interact with them in an unspecific way. The unspecific binding to the beads could be counter by saturating them (in the tube without antibody) with a neutral protein or a solution with milk (with the same idea to block sites on a nitrocellulose membrane for immunodetection) before adding to the supernatant. An other possibility is to better wash the beads after immunoprecipitation. An other method to see interaction between proteins exist, like FRET where 4

5 each proteins is marked with a fluorophore and the fluorescence changes with the distance of the markers from each other. An advantage of FRET is that it is done in vivo. Nous attestons que dans ce texte toute affirmation qui n est pas le fruit de notre réflexion personnelle est attribuée à sa source et que tout passage recopié d une autre source est en outre placé entre guillemets. [5] S Christoforidis, H M McBride, R D Burgoyne, M Zerial. The Rab5 effector EEA1 is a core component of endosome docking. Nature, 397: , 1999 February. [6] Lubert Stryer, Jeremy M.Berg, and John L. Tymoczko. Biochimie. Médecine-Science Flammarion, ème édition. [7] Uniprot. Yipf5 human. http: // Daniel Christophe Pauline Abegg Berthier Bonvin References [1] Anne Simonsen, Roger Lippe, Savvas Christoforidis, Jean-Michel Gaullier, Andreas Brech, Judy Callaghank, Ban- Hock Tohk, Carol Murphy, Marino Zerial and Harald Stenmark. EEA1 links PI(3)K function to Rab5 regulation of endosome fusion. Nature, 394: , 1998 July. [2] Carolyn A. Worby and Jack E. Dixon. Sorting out the cellular functions of sorting nexins. Nature, 3: , 2002 December. [3] J. Gruenberg. Purification de protéines exprimées dans des cellules humaines en culture, [4] Peter J. Cullen. Endosomal sorting and signalling: an emerging role for sorting nexins. Nature, 9: , 2008 July. 5