TY  - GEN
T1  - UMEPPI: An ultrasensitive detection method for protein–protein interaction
AU  - Umeyama, Mikiya
AU  - Hirose, Jun
AU  - Morohashi, Kengo
DO  - 10.17912/micropub.biology.000309
UR  - http://beta.micropublication.org/journals/biology/micropub-biology-000309/
AB  - The quantification of protein and detection of protein–protein interaction (PPI) largely requires antibody-mediated techniques such as Western blotting and immunoprecipitation. These detection methods for PPI have relatively low sensitivity compared with those for nucleic acids. Because the polymerase chain reaction (PCR) has a powerful amplification of signals derived from nucleic acids, this method for detecting nucleic acids has a higher sensitivity than general PPI detection methods. In this study, we developed a novel method for detecting PPI by converting protein signals to those of nucleic acids, which is called the ultrasensitive detection method for PPI (UMEPPI). UMEPPI uses an RNA aptamer that tightly associates with a histidine tag. The RNA aptamer is genetically fused to a gene encoding a histidine-tagged protein-of-interest (POI). Because the affinity between the RNA aptamer and the histidine peptide tag is pico mol/l (10−12 M) order as a dissociation constant (KD) (Tsuji et al., 2009), the RNA aptamer-tagged protein complex is stable through protein purification steps, and the mRNA of POI can be detected by reverse transcription PCR (RT-PCR). The goal of this study was to prove the concept of UMEPPI. The scheme of UMEPPI is shown in Figure 1A. UMEPPI uses an RNA aptamer called shot47 (Tsuji et al., 2009), which is located upstream of a coding region of the POI with a histidine peptide tag so that mRNA is tightly associated with its own protein through the histidine peptide tag. Thus, the detection of mRNA indicates the existence of shot47 and histidine-peptide-tagged POI, which is called the Hishot complex. If there is a protein interaction with the Hishot complex, the interaction can be detected through the Hishot complex mRNA by RT-PCR. It is important to evaluate the stability of the Hishot complex through the protein extraction step to detect the PPI by UMEPPI. Particularly during the extraction process, endogenous RNase in bacteria or cells may degrade RNA aptamers of the Hishot complex. To investigate the stability of the Hishot complex mRNA, we performed protein extraction with or without RNase and RNase inhibitor treatments followed by detection of the Hishot complex mRNA with quantitative RT-PCR (qRT-PCR). Figure 1B shows that the Hishot complex mRNA was stable even through the bacterial extraction steps, whereas an extra amount of RNase significantly degraded the Hishot complex mRNA. Moreover, the RNase inhibitor did not dramatically change the amount of Hishot mRNA. This suggests that the Hishot complex mRNA showed a minimal effect on endogenous RNase.
PY  - 2020
JO  - microPublication Biology
ER  -