A fusion protein for screening weak mdmx inhibitors or testing the inhibitory activity of weak mdmx inhibitors

Abstract

The invention discloses a fusion protein used for screening weak MdmX inhibitors or testing the inhibitory activity of weak MdmX inhibitors, wherein: the test sequence is the MdmX sequence or its homologous sequence; the binding sequence is the MdmX binding domain in the p53 sequence A mutant of the p53 fragment; the connecting arm sequence is a peptide segment, the upstream and downstream of which are connected to one of the test sequence and the binding sequence respectively. The tryptophan emission spectra of the fusion protein are different in the two states of binding domain and test domain, and the mutant of the p53 fragment weakens the interaction between the p53 binding domain and the MdmX binding domain compared to the wild-type p53 fragment. This function allows the MdmX inhibitor with weaker binding force to compete with the binding sequence to bind to the test sequence, thereby effectively determining the degree of inhibition of MdmX by the weak MdmX inhibitor.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the preparation and application of a recombinant protein for screening MdmX inhibitors or testing the inhibitory activity of Mdmx inhibitors, in particular to screening for weak MdmX inhibitors or testing the inhibition of weak Mdmx inhibitors The preparation and application of an active recombinant protein, more specifically relates to the preparation of a p53 polypeptide and MdmX amino-terminal fusion protein and its analogue and its application in screening MdmX inhibitors or testing the inhibitory activity of MdmX inhibitors. Background technique [0002] p53 is a tumor suppressor protein that plays a role in biological processes such as cell cycle arrest, DNA damage repair and apoptosis, and has become an important target for tumor therapy. The anti-oncogenic activity of p53 is suppressed by intracellular overexpression of MdmX and Mdm2, which is associated with more than half of ...

AI Technical Summary

Problems solved by technology

[0010] 1. When the p53 fragment and the MdmX fragment are added to the test system separately, the molar ratio of the two is difficult to determine accurately, which will bring large systematic or random errors

[0011] 2. The protocol uses fluorescein to label p53 fragments, which will make the molar ratio of fluorescein molecules and p53 fragment molecules not completely consistent in each labeling operation, which will bring large random errors and make it more difficult to carry out accurate quantitative research

[0012] 3. In the protocol, fluorescein is used to label p53 fragments, and fluorescein is easy to decompose and denature under visible light and lose its fluorescence function, which requires corresponding operations to avoid light. Photolysis will reduce the accuracy of the detection method, and requiring light protection will increase the operation The difficulty and cost of experiments

[0013] 4. Due to the hydrophobicity of fluorescein, the accuracy of detection methods and data is reduced

[0014] 5. The p53 fragment and MdmX need to be prepared separately, which will increase the complexity of the operation

[0015] 6. Although MdmX and Mdm2 are homologous proteins and their three-dimensional structures are highly similar, the existing Mdm2 small molecule inhibitors have very weak inhibitory effect on MdmX

[0016] 7. The MdmX inhibitors with significantly different acting forces are all tested with the same model, and it is impossible to distinguish strong MdmX inhibitors from weak MdmX inhibitors. More importantly, weaker MdmX inhibitors may be due to ineffective Competitive binding to the p53 domain without detection

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