Study On Expression And Purification Conditions Of Short Peptides Of PTEN Targeted Degradation

How to repair after the damage of the central nervous system has been receiving much attention.In recent years,the studies have shown that the ability of neuronal axons to regenerate is a key factor affecting the repair of neuronal damage.The results confirmed that the inhibition of PTEN gene activation of m TOR can promote the regeneration of injured axons.Therefore,how to silence PTEN to activate m TOR is particularly important.In this paper,by synthesizing PTEN to degrade short peptides,the TAT-PDZ-CTM protein was expressed in the prokaryotic expression system,and purified by different chromatographic principles.The purification conditions for obtaining high-purity proteins were screened for further application of targeted PTEN-degrading proteins.The m TOR pathway is activated to study the regeneration mechanism of injured neuron axons to provide high purity proteins.Method:1.Constructing TAT-PDZ-CTM sequences using molecular biology methods.2.The protein TAT-PDZ-CTM was expressed by the prokaryotic expression system(E.coli BL21 competent cells),and high content of protein was successfully expressed under the effect of IPTG-induced expression.SDS-PAGE and Western blot were used to determine whether the expressed protein was correct.3.By comparing the two purification steps,respectively,the purification step 1: S75 gel chromatography-ultrafiltration desalting and purification,step 2: composite cation chromatography-G25 gel is desalted to treat the protein,and finally the purity of the purified sample is identified by SDS-PAGE and HPLC detection,and the appropriate purification method and conditions are finally determined.4.The activition of TAT-PDZ-CTM was verified by Western blot assay in a neuron-like SH-SY5 Y cell assay.Result:1.Successfully constructed a recombinant expression vector p ET28a-TAT-PDZ-CTM2.Through the expression system of E.coli,the expression of TAT-PDZ-CTM was expressed by using IPTG to induce expression.3.For the 6*His tag contained in the N-terminus of the sequence design,the specific selection is purified by affinity chromatography to effectively purify the protein.4.The purity of the obtained protein was 94.31% and the content was 0.27 mg/ml by the first purification step: S75 gel chromatography-ultrafiltration desalting;complex cation chromatography-G25 gel demineralization gave a protein purity of 98.57% and a content of 1.20 mg/ml.5.In normal cells SY5 Y,35 mg/ml TAT-PDZ-CTM protein was added in proportion,and the intracellular PTEN was effectively degraded by Western Blot.Conclusion:1.By comparing the two purification methods,the purification method 2 is determined: Ni affinity chromatography-composite cation chromatography-G25 gel desalination method and conditions than purification method 1: Ni affinity chromatography-S75 gel chromatography-ultrafiltration desalting condition is used to purify the protein TAT-PDZ-CTM,and the final sample obtained has higher purity and content.Therefore,the purification method 2 is more suitable for the purification of TAT-PDZ-CTM.2.The short peptide TAT-PDZ-CTM,which degrades the endogenous protein PTEN,was successfully expressed.