| Leukemia is a malignant disease of the hematopoietic system with high incidence and death rate. While the number of incidence keeps rising, great attention is attracted to the molecular mechanism of this malignancy. Ikaros family, a group of critical transcription factors mostly restricted in hematopoietic system, has been shown to regulate several important genes for hematopoiesis, and displays significant influence on the development of blood cells, especially the lymphoid cells. Aberrant expression or dysfunction of these proteins has a close relationship with hematopoietic malignant disease. Ikaros family members are characterized with two sets of C2H2-type zinc-fingers:four zinc-fingers at N-terminus recognize a specific DNA sequence, while another two fingers at C-terminus are found to mediate protein-protein interactions and to regulate the activity of N-terminal region and the life-span of the protein. Ikaros members can either activate or repress the expression of target genes in three levels, chromosome level, nucleosome level and RNA polymerase II level, by means of direct association with the regulatory elements of target genes, recruitment of chromatin remodeling complexes, or recuitment of target genes into pericentromeric heterochromatin domain. C-terminal region of Ikaros is required to achieve the transcriptional regulation by mediating the interactions with other proteins or homodimerization. In this study, highly conserved C-terminal regions of Ikaros/Aiolos have been taken as the research object, and the experiments of molecular cloning, protein over-expression, protein purification, and crystallization have been performed. The results are following:1. Construction of various expression plasmids are achieved through molecular cloning. MBP fusion proteins with different linkers vary in the efficiency of protease digestion; 2. MBP-IkarosC/AiolosC fusion proteins in high purity are obtained by using of several chromatography strategies, based on the differences in the affinity of protein tag, surface charge, hydrophobicity, molecular weight and shape; 3. Experiments confirm that the C-terminal part of Ikaros/Aiolos is able to interact with the supporting base of chromatography beads, which causes the difficulty in elusion; 4. Several potential conditions to grow protein crystals are detected. Preliminary optimization tests have shown that the conditions were of good repeatability. Optimization experiments are still in progress.In another part, I have done some research on TREK1, which is a subtype of two-pore domain K+(K2P) channels, widely expressed in central and peripheral nervous system. It is a robustly mechanosensitive channel, and is modulated by chemical and physical stimuli including lipids, phosphorylation, temperature, pH and many other factors. It plays an important role in physiological functions related to neuroprotection, pain, anaesthesia and depression. TREK1 is characterized by four transmembranes helices(M1～M4),2 pore domains(2P), a short N terminal sequence and a long C terminal region at the cytoplasmic side. Regulation of TREK 1 activity by various stimuli has been partly ascribed to a C-terminal region right after the fourth transmembrane (M4) helix. My study has been focussed on the C terminal region of TREK 1, with the experiments of molecular cloning, protein purification and crystal screening. Vectors of pET28a, pET30a, pMAL, pFN18a have been selected to carry various truncated fragments of TREK1C coding gene, and 11 expression plasmids have been constructed. Through test expressions, the plasmid of pMAL-TREKIC has been selected for its better yield of the fusion protein in supernatant. Subsequently, MBP-TREK1C was purified by MBP affinity chromatography and gel filtration chromatography to finally reach a high purity. Crystallization conditions of MBP-TREK1C were screened with Index and Wizard kits, and the examination was to be carried on. |
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