Polypeptide for inducing protein degradation and application thereof
A protein degradation and protein technology, applied in the direction of peptide/protein components, fusion polypeptides, fusion with degradation motifs, etc., can solve the problem of lack of peptides, and achieve the effect of inhibiting degradation
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Embodiment 1
[0054] Example 1: Construction of target protein and degradation protein fusion plasmid
[0055] Fusion expressing a specific polypeptide sequence and target protein to construct a plasmid for fusion expression. After sequencing and identification, the plasmid was transfected into HEK293 host cells; after plasmid transformation, observed under a fluorescent microscope, the successfully transfected cells showed red fluorescence.
[0056] Specific steps are as follows:
[0057] In this example, there are two ways of linking the polypeptide to the target protein,
[0058] 1. The plasmid in this example is pcDNA3.1, the mCherry protein is used as a reporter gene, and the N-terminus is mCherry tandem p2A self-shearing isolated polypeptide, followed by GFP-protein degradation-inducing polypeptide (abbreviated as GFP-degradation-inducing polypeptide, specific example is SEQ ID NO: 1).
[0059] 2. The plasmid in this example is pcDNA3.1, the GFP protein is used as a reporter gene, t...
Embodiment 2
[0066] Example 2: Fluorescence detection for target protein
[0067] In this example, after 24 hours, the cells in Example 1 were collected, placed under an inverted fluorescence microscope, and the green fluorescent protein was observed with a fluorescence excitation wavelength of 488nm, and the fluorescence signal of mcherry was observed with a fluorescence excitation wavelength of 520nm. The control plasmid was mCherry-p2A tandem GFP without protein-inducing degradation polypeptide.
[0068] Experimental results: The cells introduced with the engineered plasmid showed reporter gene fluorescence under a fluorescent microscope, indicating that the recombinant plasmid was successfully introduced into the host cell, and the fluorescent signal of the protein in series with the degraded protein was attenuated.
[0069] figure 2 The results indicated that whether SEQ ID NO:1 is linked at the N-terminus or the C-terminus, it can effectively degrade the fusion protein. image 3 T...
Embodiment 3
[0073] Example 3: Fluorescence detection for target protein
[0074] In this example, the cells in Example 1 were used, a proteasome inhibitor (MG132) was added to the culture medium, 2.5 μM MG132 was added, and after 12 hours, 24 hours and 38 hours, the Use the fluorescence excitation wavelength of 520nm to observe the green fluorescent protein, and use the fluorescence excitation wavelength of 520nm to observe the fluorescence signal of mcherry. The control plasmid was mCherry-p2A tandem GFP without protein-inducing degradation polypeptide.
[0075] Figure 5 The results indicated that the inhibitor MG132 could block the peptide-induced protein degradation, and it increased with time.
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