Study On Cell Delivery Of Tumor Suppressor Proteins Sufu And P53 And Crystallization Of β-monooxygenases Of Bacterial Pyridine Ring

Hedgehog signaling pathway plays an important role in the development of vertebrates and invertebrates.In humans,the disorder of Hedgehog signaling pathway is closely related to neonatal birth defects and tumorigenesis.The Suppressor of fused（Sufu）protein in the Hedgehog signaling pathway blocks Gli/Ci in the cytoplasm to inhibit Gli/Ci regulation of target gene transcription.In this study,a green fluorescent protein GFP-labeled Sufu protein expression vector was constructed,and His-GFP-Sufu protein was successfully expressed in Escherichia coli,and His-GFP-Sufu with high purity protein was purified by various chromatographic means.We used nanomaterials to deliver His-GFP-Sufu protein into mammalian cells,and used luciferase assay and other methods to conduct preliminary research on the function of His-GFP-Sufu protein delivered into mammalian cells.This laid the foundation for the development of the tumor suppressor protein Sufu as an anti-cancer protein drug.p53 protein is an important tumor suppressor protein.p53 protein plays an important role in regulating cell proliferation and cancer development.The p53 protein can block the cell cycle,initiate programmed death of abnormal cells,stabilize the cell’s genome,repair damage to DNA,and reduce the incidence of cancer.In this study,full-length p53 protein was expressed in Escherichia coli,and p53-linker-m Cherry protein with high purity and red fluorescent protein m Cherry tag was successfully purified by Ni²⁺-NTA affinity chromatography and gel filtration chromatography.We also used MBP column affinity chromatography,TEV enzyme digestion of MBP tags,on-column digestion,ion exchange chromatography,gel filtration chromatography and other means to purify the p53 protein with the green fluorescent protein GFP tag.These studies laid the foundation for the subsequent delivery of p53 protein into cancer cells to inhibit cancer cell proliferation.The 6-hydroxy-3-succinylpyridine（HSP）monooxygenases HspA,HspB of Pseudomonas putida S16 strain and the 6-hydroxynicotinic acid monooxygenase NicC of the KT2440 strain belong to the pyridine ringβ-monooxygenase.They play an important role in the microbial degradation of pyridine heterocyclic compounds.In this study,high-purity HspB and NicC proteins were expressed and purified in Escherichia coli.We obtained high-quality single crystals of HspB/Se Met-HspB,NicC/Se Met-NicC,and co-crystals of 6-hydroxynicotinic acid and NicC,and collected X-ray diffraction crystal data at Shanghai Synchrotron Radiation facility to finally resolve the NicC single crystal structure.This study has laid a foundation for explaining the reaction mechanism ofβ-hydroxylation of pyridine heterocyclic compounds,and is of great significance for the study of microbial metabolism of pyridine heterocyclic compounds.