Bait Expression Assay

The last control experiment aimed to confirm whether EYS bait fragments were expressed in yeast cells. To do that, protein extracts were prepared from transformed Y2HGold strains and Western blot analysis was performed. In order to prepare the extracts, single colonies of transformed Y2HGold strains were inoculated in liquid selective medium (SDO/-W). Cultures transformed with wild type pGBKT7 and pBD-DEST-WT vectors were used as positive controls whereas wild type Y2HGold yeast strain was used as a negative control. Inoculated colonies were not older than four days, otherwise the extraction of proteins would have been hindered due to thickening of the yeast cell wall. The cultures were kept in the log phase of growth, which was necessary to enable expression of the bait fragments. In the late log phase, the expression of bait fragments could have been impeded by the increasing concentration of ethanol that accumulates in the culture medium as a by-product of yeast metabolism. The manual provided by Clontech recommends two extraction methods, Urea/SDS method and TCA method. Both of the methods were tested, however, bands corresponding to the bait fragments were not observed in Western blotting.

The methods recommend by Clontech involve many steps and disruption of yeast cells is achieved by vortexing the cultures with glass beads. These generally time consuming and rather harsh procedures could have had an impact on degradation of the proteins of interest during extraction. Therefore, another reagent enabling extraction of proteins from yeast, named Y-Per Plus (Thermo Scientific, USA) was used. Y-Per Plus is a detergent based lysis buffer allowing fast extraction of yeast proteins that is carried out at room temperature and the use of glass beads is not required. In order to obtain as high concentration of proteins as possible, only 40 µl of extract were prepared from each of the 5 ml overnight cultures. The concentrations obtained were checked using a BCA assay and the extracts were resolved by SDS-PAGE, transferred to PVDF membranes and probed with anti GAL4 DBD antibody. The developed membranes are shown in Figure 3.7.

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Figure 3.7 Western blot analysis of the expression of EYS bait fragments in Y2HGold yeast

cells. Extracts were prepared using Y-Per plus reagent and resolved on pre-cast 4-20 % gradient gels. Loading was optimised using BCA assay and approximately 40 µg of total protein extract were loaded in each well. Immunoblotting (IB) was performed with using anti-GAL4 DBD rabbit antibody and the secondary antibody was HRP-conjugated goat anti-rabbit. Bio-Rad Precision Plus Protein Standard was used to assess the size of protein bands. Samples 1-13 represent different samples run in the experiment and the expected size of each protein band is given in brackets. The red arrow indicates the band of wild type GAL4 DBD fused with 132-236 amino acids of wild type fragment C of lambda cI repressor (positive control 2).

Identification of Novel Interacting Partners of EYS

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There were two positive control samples, each of which contained GAL4 DBD wild type. In the first positive control GAL4 DBD wild type was expressed from the pGBKT7 wild type vector (lane 1) and in the second, a pBD-DEST-WT vector was used (lane 13). The band obtained from the latter appeared higher as this control vector encodes GAL4 DBD fused with 132-236 amino acids of wild type fragment C of lambda cI repressor. As it can be observed, there is a clear intense band in lane 1, but a weaker band was detected in lane 13 (marked with red asterisk). The presence of bands in the positive controls proves that the protein extraction was effective and that the conditions of SDS-PAGE and Western blotting were properly optimised. Clear bands were not, however, seen for any of the bait fragment proteins. Upon in-depth analysis of blots, it can be noticed that, in the lanes representing EYS bait fragments, there is a smeared signal in the range of approximately 75-350 kDa. The smeared signal suggests that the proteins in this range could have degraded during extraction, sample processing or gel separation. One could argue that degradation should not have been an issue since the wild type GAL4 DBD did not give a smeared signal. It should be pointed out, however, that there are two bands visible in lane 1, one at the size of wild type GAL4 DBD (~22 kDa) and the second one at about ~12 kDa. Even though the signal was not smeared, the presence of the second band implies that some level of degradation had happened and resulted in the appearance of two protein bands. Furthermore, it is necessary to highlight that the sizes of EYS bait fragments are relatively large and, therefore, the expected bands would have been detected at a lot higher molecular weights than wild type GAL4 DBD. It is also natural that the extraction of higher molecular weight proteins is more challenging and a lot more liable to potential sample degradation. The size of bait fragments may have had an impact on the efficiency of expression in yeast cells and another scenario could be that the expression level of bait fragments was not sufficient enough for the Western blotting to detect the protein.

Distinctly visible protein bands of the bait fragments were not detected and therefore, the important question of whether the baits were expressed in Y2HGold yeast cells could not be clearly answered. However, the baits passed the previous control experiments proving that the expression of proteins from the bait vectors was taking place – otherwise cultures would not have been able to grow on selective media lacking crucial amino acids that were encoded on the bait vectors

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for the purpose of selection. Based on that, it was concluded that the EYS bait fragments were suitable for Y2H screening.