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) Inhibition of the 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.
2-2)
こんにちは3
こんにちは4
Reference
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