Purification and Crystallization Studies of Transcription Factor CCAAT/Enhancer Binding Protein delta and Inflammatory Factor Long Pentraxin 3

Abstract

Abstract Part A: CEBPδ Transcription factor CEBPδ is involved in cell differentiation motility growth arrest proliferation and cell death Studies also suggested that CEBPδ functions differed depending on cell type and cellular context CEBPδ has been proved as a tumor suppressor in some cancers for example in breast cancer But CEBPδ also help glioblastoma cell’s survival by inflammation response and induce metastasis Previous studies had reported CEBPδ has many posttranslational modification sites and need to form a dimer with other family member or different proteins for its function But the detailed mechanism and function of these modifications and dimerization are still unknown In our research we try to study and explain these mechanisms by crystallography and X-ray diffraction So we made constructs including transactivation domain (5-186) bZip domain (190-270) and full length (5-270) of CEBPδ， and all these three proteins can be over expressed We can’t get enough full length to crystallization because it’s poor binding affinity Transactivation domain could be pulled down enough but it contained nonspecific bond Finally we use ion exchange and gain pure protein and screening them for potential condition of crystallization Once optimize the potential condition it may form a crystal and provide more detail structure conformation According to size-exclusion chromatography data we found that CEBPδ without its bZIP domain still formed a homodimer We supposed that besides bZIP domain the N-terminus contributed a physical interaction to dimerization Part B PTX3 PTX3 has been considered as an inflammatory marker because PTX3 can recognize the extracellular pathogens and induce inflammation response for immunity And it interacts with many cytokines within its N-terminus or C-terminus When PTX3 deposited onto apoptotic cell it recruited the C3 and C4 on apoptotic cell This process presents a “eat me” signal for macrophage But the fluid phase PTX3 would inhibit the C3 deposition for an anti-apoptotic function Although the SAXS data of PTX3 was reported the mechanism of these functions can’t be solved so it need structure study to provide more information In our study we made construct of full length (19-381) N-terminal domain (19-182) and C-terminal CRP like domain (180-381) of PTX3 The result of expression presented that the N-terminus from bacterias was soluble the same as form eukaryotic cell but C-terminus and full length were insoluble By purification experiment data we supposed that the PTX3 N-terminus form a tetramer in solution like eukaryotic PTX3 and there was degradation which we still try to remove Although the N-terminal recombinant protein had degraded it was stable so we still screened this protein with degradation and try optimization for crystal which can provide more detail information of PTX3 mechanism The full length and C-terminus formed inclusion body We used Guanidinium-HCl to dissolve the inclusion body and used dialysis for gaining soluble protein But the protein production was too low to crystallization According to size-exclusion chromatography data of protein purified by dialysis we suppose that the C-terminus form a dimer the same as in eukaryotic cell but full length did not form a octmer The different between prokaryotic and eukaryotic modification system could be a reason of this result

Date of Award	2014 Feb 26
Original language	English
Supervisor	Yu-Chih Lo (Supervisor)