Fusion protein for screening weak MdmX inhibitor or testing inhibition activity of weak MdmX inhibitor

Abstract

The invention discloses a fusion protein for screening a weak MdmX inhibitor or testing the inhibition activity of a weak MdmX inhibitor. The testing sequence is an MdmX sequence or a homologous sequence thereof. The combination sequence is the mutant of p53 segment of the MdmX binding domain of p53 sequence. The linker arm sequence is a peptide segment, and the upstream and lower stream of the linker arm sequence are connected to one of the testing sequence and the combination sequence. The emission spectrums of tryptophan in the fusion protein are different when the combination structural domain and the testing structural domain are connected / disconnected; and for the mutant of p53 segment, compared with a wild p53 segment, the interaction between the p53 combination structural domain and the MdmX combination structural domain is weakened, so that the MdmX inhibitor with a weak binding force can compete with the combination sequence to combine the testing sequence, and thus the inhibition degree of the weak MdmX inhibitor on MdmX can be effectively measured.

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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