Study Of Preparation Of Segmental Isotopic Labeling ApoE With Intein-mediated Protein Ligation And Structure Of RAP

ApoE is one kind of apolipoprotein of 299 amino acids. It can bind to LDL receptor protein, lipid and heparin. It plays important roles in modulating the metabolism of lipid and associated to the type III hyperlipoproteinemia, atherosclerosis, and Alzheimer's disease (AD). The understanding of the structure and molecular biological function of apoE is important and will help us to prevent and cure those diseases. The structure of apoE N-terminal (apoE1-199) has been solved with X-Ray, apoE C-terminal and full-length apoE are still unknown. ApoE is too big to solve the structure by NMR, so we try to prepare segmental isotopic labeling apoE for NMR study. Receptor-associated protein (RAP) is a chaperon protein of LDL receptor protein, it is consist of 323 amino acid residues. It can bind to the immature LDL receptor and help it refold correctly. In this paper, we study the methodology of preparing the full-length apoE by protein ligation and some property of full length RAP and some domain of RAP.In this paper, different ways were tried to prepare the alpha-Cys C-terminal, alpha-thioester N-terminal and carry out ligation. Three ways were tried to prepare alpha-Cys c-terminal: directly to expression, CNBr and Factor was use to cut fusion protein. After introducing reducing agent BME, factor Xa cleavage was found suitable to achieve this goal. To prepare alpha-thioester, apoE N-terminal was subcloning into IMPACT expression system. It is first time to observe that the high yield of protein can be gotten from basic medium, it also be found most protein contain alpha-thioester after thiolysis are easily hydrolysis to carboxyl acid which can't be used in protein ligation. Different ways were tried to carry out the protein ligation, Intein-mediated protein ligation was found to be the best way to get ligation product. Finally CL-6B heparin column was used to purify to product. Segmental isotopic labeling apoE were prepared in large scale for NMR study.In order to block the autocleavage of fusion protein in pTWIN1 vector during expression, two mutants were successfully design base on protein engineering. RAP was also studied with cross-linking, CD and lipid binding assay.