MU: Data curation, Investigation, Methodology, Formal analysis
JH: Data curation, Investigation
KM: Writing - original draft, Writing - review and editing, Supervision, Funding acquisition, Conceptualization
An ultrasensitive detection method for protein–protein interaction. (A) A schematic representation of the UMEPPI method. Shot47 is a ribonucleic acid (RNA) aptamer that strongly associates with a histidine tag. The shot47 complex and protein-of-interest is called the Hishot complex. The interaction can be detected by performing reverse transcription polymerase chain reaction (RT-PCR) or quantitative RT-PCR (qRT-PCR) of purified mRNA of the Hishot complex. (B) The stability of mRNA in the Hishot complex. The Hishot complex was extracted from the expression of the host
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 (
For the evaluation of UMEPPI, we used a known combination of PPI that we previously reported (Umeyama and Morohashi, 2020). GLABROUS1 (GL1; At3g27920), PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1; At1g56650), and GLABRA3 (GL3; At5g41315) are transcription factors involved in organ development and metabolite biosynthesis in seed plant,
There are various detection methods of PPI that do not require antibodies (e.g., surface plasmon resonance, quartz crystal microbalance, mass spectrometry, the phage display system, and the yeast two hybrid system) (Braun and Gingras, 2012; Rao
A total of 50 µM of oligo DNA fragments, UM_5′Xba1_shot47 (5′-CTAGAGGTATATTGGCGCCTTCGTGGAATGTCAGTGCCT-3′) and UM_3′Xba1_shot47 (5′-CTAGAGGCACTGACATTCCACGAAGGCGCCAATATACCT-3′), were mixed in 10 µL of TE (10 mM Tric-HCl pH8.0 and 1 mM EDTA pH8.0). Then, the oligo fragments were heated at 95°C using a thermal cycler. After 5 min, the thermal cycler was turned off, and the fragments were left overnight to reach room temperature. The pET21 vector was treated with XbaI restriction enzyme at 37°C for 1 h. After ethanol precipitation, the digested pET21 and shot47 dsDNA fragments were ligated using a DNA ligation kit <Mighty mix> (TAKARA Bio, Shiga, Japan) at 16°C for 30 min.
The coding sequence (CDS) of GL1 (At3g27920) was amplified with MU_pET_GL1_F (5′-AATGGGTCGCGGATCCGAAATGAGAATAAGGAGAAGAGATG-3′) and MU_pET_GL1_R (5′-TGTCGACGGAGCTCGAATTAAGGCAGTACTCAACATCACC-3′). The amplified GL1 CDS fragment was inserted into pSpET using HiFi DNA Assembly Master Mix (NEB, NEB, Ipswich, MA). The pSpET-GL1 was transformed into
The DNA fragments of PAP1 (At1g56650) and GL3 (At5g41315) were cleaved by
The induced culture was collected in a 500-mL bottle followed by centrifugation at 3,500 rpm for 15 min at 4°C After discarding the supernatant, 30 mL of phosphate-buffered saline (PBS) (1,370 mM NaCl, 27 mM KCl, 81 mM Na 2 HPO 4 ·12H 2 O, and 14.7 mM KH 2 PO 4 ) was added, resuspended, and centrifuged at 3,500 rpm for 15 min at 4°C.
Lysozyme was added in 10 mL of the bacteria-expressed GL1-Hishot protein to achieve 1 mg/mL as a final concentration. In the case of the RNase or RNase inhibitor treatment, RNase I was added to achieve 10 ng/mL as a final concentration, or RNase inhibitor (SUPERase In; Thermo Fisher Scientific, Waltham, MA) was added to achieve 0.8 U/µL as a final concentration, respectively. Then, the reaction solution was incubated for 30 min at 37°C followed by sonication on ice until the stickiness was gone. The sonicated solution was centrifuged at 9,000 rpm for 15 min at 4°C. Then, 500 µL of the supernatant was transferred into a tube that contained 100 µL of the host protein. Next, 300 µL of a 30% slurry of glutathione sepharose 4B was added in the tube and incubated at 4°C with rotation overnight.
The reaction extract containing GL1-Hishot proteins and GST proteins was centrifuged at 500 rpm for 10 min at 4°C, and the supernatant was removed. Then, the beads were washed three times with 500 μL of PBS. Next, 350 μL of TRI Reagent (Cosmo Bio, Tokyo, Japan) was added into the beads. The upper layer was transferred into a new tube, and RNase-free DNase I (Sigma-Aldrich, St. Louis, MO) was added to achieve 0.1 U/µL as a final concentration. Then, the RNA was purified by phenol–chloroform extraction followed by ethanol precipitation. The purified RNA was subject to reverse transcription using a ReverTra Ace qPCR RT kit (Toyobo, New York, NY) in a 20-µL reaction volume according to the manufacturer’s protocol.
For semi-qRT-PCR, MU_GL1_seq (5′-GTTCATCACTGCCGCCACAC-3′) and T7 terminator primer (5’-GCTAGTTATTGCTCAGCGG-3’) primer sets were used. After, 25, 30, and 35 cycles of PCR, the reaction was subjected to electrophoresis with 2% agarose gels. For qRT-PCR, the primer sets of YT_GL1_qrt_F (5′-CCGCCACACCTTCTTCTTGTCATC-3′) and YT_GL1_qrt_R (5′-ATACGACGCCGTTAAAGCTCTTGG-3′) were used.
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This work was supported by Grant-in-Aid for Scientific Research on Innovative Areas “Frontier Research on Chemical Communications” Grant Number 18H04631.