Team:Kyoto/Result
In starting the experiment, we conducted an experiment demonstrating how the salt concentration of the solution actually affects the protein-protein interaction by using the model protein. In this example, we chose GFP(BBa_E0040), which is commonly used in many experiments as a model protein.
TDH3 promoter and CYC1 terminator were added to both ends of ORF and cloned into pRS316 which is a shuttle vector of S. cerevisiae and E. coli. The resulting plasmid was transformed into wild-type yeast strain BY4741 to overexpress GFP in yeast. As a comparative experiment, yeast expressing RFP(BBa_E0010) with the same set of promoter and terminator was used.
First, photographs of pellets recovered from the culture medium of yeast cells used in this experiment are shown.
Figure1. A picture of yeast expresses RFP or GFP
As can be easily seen, the yeast pellet overexpressing GFP was colored in a pale yellow color while the yeast overexpressing RFP was colored in a thin red color. From this, it was confirmed that both GFP of BBa_E0040 and RFP of BBa_E0010 can be expressed in large amounts in yeast cells without changing the codon and that the expression level thereof is so large as to be visually observed under visible light without breaking the yeast.
In order to investigate yeast proteins interacting with GFP, an experiment was conducted to purify expressed GFP protein from yeast lysate using immunoprecipitation. Anti-GFP nanobody (GST fusion protein on Glutathione sepharose beads) was used for sedimentation, and the obtained precipitate was visualized by SDS-PAGE and subsequent Silver Stain.
This yeast was placed in a mortar, liquid nitrogenized and crushed, and a lysate was prepared with a buffer having a salt concentration of zero. The color from the fluorescent protein was clearly transferred to the supernatant by this treatment in both proteins. Figure2 shows the results of electrophoresis of GFP pull down after adding Sepharose bead conjugated with anti-GFP nanobody to this lysate.
Figure2. Immunoprecipitation of GFP from Yeast lysate
As shown in Figure2, the band of ① is GST-GFPnb, and the band of ② (5-8) is GFP. GFP is clearly immunoprecipitated by GST-GFPnb. Furthermore, since the band intensities of ① and ② are equal, it is understood that GFP and GFPnanobody are linked at about 1: 1. This indicates that the binding between this GFP-GFPnanobody is strong and GFP is sufficiently contained in the lysate.
In the band of ③, bands are seen at low salt concentration, and it can be confirmed that the band becomes thinner as the salt concentration becomes higher. This is thought to be due to the nonspecific interaction between nanobody and the protein contained in the lysate under low salt concentration. In addition, the band of ④ of 8 lanes is a band that can be seen only at 1000 mM and in the presence of GFP. This is thought to be due to the interaction between GFP and protein in lysate generated under high salt concentration condition. These suggest that salt concentration can influence the three-dimensional structure of protein and its interaction.
From next section, we will show how to prepare yeast which adjusts salt concentration in solution.
2-1) To inhibit Na+ efflux
In order to make yeast that effectively intake the salt from the environment, we aimed to reconstruct in yeast Na+-isolation system of salt-resistant plants, which sequester Na+ into their vacuoles. We first tried to knockout NHA1 and ENA1, the critical components of Na+ efflux of budding yeast.
Since there is no ENA1 knockout strain in the Yeast Knockout Collection, we amplified hygromycin resistance cassette from pFA6a-hphMX6 plasmid and tried to knockout genomic ENA1 gene utilizing homologous recombination. We transformed PCR products into wild type yeast and obtained hygromycin-resistant colonies. Genomic PCR from obtained colonies successfully identified the strains that have desired mutation. Using the same method, we successfully made ΔENA1ΔNHA1 strain from ΔNHA1 strain.
Moreover, we asked Dr. Uozumi in Tohoku University to provide us with G19 strain that lacks all of ENA1 family genes(W303 yeasts have a cluster comprised of ENA1-ENA2-ENA3-ENA4). Although he kindly gave us G19 strain, we also introduced ENA1,2,5 knockout strain, (they are equivalents of ENA1-4 in W303 yeasts) to ΔNHA1 strain, due to the limited repertoire of selection markers that G19 strain has.
Finally, we got ΔNHA1 / ΔENA1ΔNHA1 / ΔENA1 / ΔENA1,2,5.
All genes we disrupted so far encode Na+ or K+ transporters that are responsible for Na+ export from cytoplasm.
Therefore, disruption of any of these genes would lead to decreased export efficiency of excessive salt. Some researchers reported that yeast that lacks these genes showed increased NaCl-sensitivity. To compare salt-resistance of knockout strains we prepared, we performed in vivo spotting assay.
Figure3. Spotting assay of KO yeast strains
Figure3 shows representative results of spotting assay. With 200 mM NaCl in YPD, wild type strain formed large colonies after 2 days of incubation. In contrast, G19 strain and ΔENA1,2,5ΔNHA1 showed little or no colonies, indicating dramatical increase of salt-sensitivities of these strains. Under the same condition, Zygosaccharomyces rouxii, an yeast strain known to show high salt-resistance (as they have been used to make soy sause in Japan), made large colonies. This results encouraged us to integrate genes of Z. rouxii to our device.
2-2）To increase salt-resistance of yeast
The result we described above indicate that we successfully disrupted proper genes for our purpose, and thus mutants could not export Na+ from their cytoplasm, which is favorable to make salt-absorbing yeasts. However, from these results, we also noticed serious problem that our project has.
As long as we use the mutants that absorb salt, we have to face the problem of salt-sensitivity that our device has. If we could sequester Na+ in the vacuole of yeast as we initially planned, salt-resistance might be recovered. However, even the yeast equipped with Na+ sequestration system would suffer from high salt condition to some extent. Hence, we concluded that we have to give yeast salt-resistance to overcome this problem.
In order to make salt-resistant yeast, we took three approaches; to sequester Na+ into vacuole by active transportation as we initially planned; to prepare compatible solutes to mitigate cell stress caused by osmotic pressure; and to minimize the alteration of the higher order structures of proteins, which is caused by high Na+ concentration, by efficiently expressing chaperons.
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Reference
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