The Expression, Purification And Crystallization Of Trypanosoma Brucei EIF4E

This thesis contains two parts: the recombinant protein expression of Trypanosoma brucei eIF4E and the crystal structure research of Zebrafish MO25.Trypanosoma brucei, which belongs to Kinetoplastida, Division of trypanosome Trypanosoma, is a pathogenic parasite with serious infection. In human, this worm causes sleeping sickness, Chagas disease and leishmaniasis, mainly spread through tsetse flies in Africa. The worms cause great risk of the health of human and other mammals. It is still a big chanllege for the development of the effective vaccines for these parasites. The only clinical treatment for these dieases was chemotherapy. The key to identify a new therapeutic direction and resolve the drug resistance problems was to solve the structure and find the new targeted drugs.Eukaryotic translation initiation factor4E, with25KD molecuLar weight, mainly distributed in the cytoplasm. The role of eIF4E in translation was to reguLate the cap-dependent structure and bind with the5’-mRNA. The reguLation of eIF4E was through the binding of the scaffold protein eIF4G and the activity of4E binding proteins. Both eIF4G and4EBPs will increase the binding affinity of eIF4E and the5’ end mRNA. The eukaryotic translation initiation factor eIF4E (TbeIF4E) contains4subtypes in Trypanosoma brucei, with different functions each.It is still not clear the process of eIF4E combinate with other proteins and the reguLation of the translation. In the present study, the plasmids which express Trypanosoma brucei eIF4E1(1-233aa)-C-His-pET22b, eIF4E2(1-251aa)-C-His-pET22b, eIF4E3(1-442aa)-C-His-pET28a, eIF4E4(1-427aa)-C-His-pET22b were built. The expression testes proved that only eIF4E1was soluble protein. After the purification by Ni-NTA affinity chromatography and the Superdex200gel filtration chromatography process, the resuLts of SDS-PAGE showed that the protein we collected was high of purity and good in uniform. Then the sitting drop vapor diffusion method was used to grow the protein crystal.MO25, a conserved scaffold protein, activates the tumour suppressor LKB1with the pseudokinase STRAD. MO25also promotes the activities of the STE20-family kinases MST3, MST4, STK25, SPAK and OSR1. Zebrafish MO25was purified and crystallized, and a crystal of zebrafish MO25diffracted to2.9A. resolution and belonged to space group P3221, with unit-cell parameters a=b=156.665, c=221.251A. The structure of zebrafish MO25was determined by molecuLar replacement. It is constituted of seven helical repeats. Structural comparison indicates that the overall structures of zebrafish and human MO25are very similar, suggesting that MO25has conserved functions in zebrafish. This work provides a structural basis for further functional and evolutionary studies of MO25.