翁啟惠Wong, Chi-Huei臺灣大學:生化科學研究所黃劭軒Huang, Shao-HsuanShao-HsuanHuang2010-05-042018-07-062010-05-042018-07-062008U0001-1107200815051400http://ntur.lib.ntu.edu.tw//handle/246246/178808Bcl-2家族蛋白在程序性細胞死亡過程中扮演相當重要的角色。Bcl-2 蛋白家族主要可分為兩大類。一類為Pro-apoptotic，另一類則為Anti-apoptotic。Bik 屬於前者，而Bcl-XL屬於後者。他們在細胞中互相拮抗，並調控細胞凋亡的程序。去的研究發現，Bik 可被CKII（Casein Kinase 2）磷酸化。為了要模擬此磷酸化過程，洪明奇院士團隊將Bik上之Thr33和Ser35位置定點突變成Aspartate，並將此突變株取名為BikDD。在試管試驗中，BikDD 增強了其原本對Bcl-XL之親和力，另外在不同之癌症細胞株（乳癌、前列腺癌、胰臟癌、肺癌），BikDD 皆展現其增強之毒殺效果。最近他們設計了一種只會針對胰臟癌表現的啟動子（CCKAR-VISA），利用其專一性，BikDD 蛋白特定地表現在帶有胰臟癌的老鼠身上，並根除了胰臟腫瘤， 此一成果提供了未來基因療法用於人體試驗的可能性。篇論文的目標為研究BikDD和Bcl-XL複合體，希望透過對結構之瞭解，提供更多資訊給BikDD基因療法用於人體試驗基礎，以及癌症新藥的開發。BikDD和Bcl-XL皆屬於膜蛋白，因其疏水物理特性，增加了純化及長晶的困難度。我們首先利用大腸桿菌大量表現出蛋白複合體，再利用不同的純化方法，將此複合體從水溶液狀態或膜上純化出來。為了要拿到符合長晶條件的樣品，我們亦測試了不同之介面活性劑，不同片段之表現，以及Bicelle 結晶法，並利用DLS和AUC來驗證其均質度。在拿到夠均質的樣品之後，我們進行了結晶條件測試。外，我們也利用FRET技術，設計了一種可用來篩選Bcl-XL抑制物的平台，希望藉此找出癌症藥物前驅物，提供治療癌症的契機。Bik and Bcl-XL are Bcl-2 family members. They play crucial roles in the regulation of programmed cell death. Bcl-2 family can be broadly categorized into two groups. One is pro-apoptotic proteins, the other is anti-apoptotic proteins. Pro-apoptotic and anti-apoptotic proteins antagonize each other and mediate apoptosis. Bik belongs to the pro-apoptic type , Bcl-XL belongs to the anti-apoptotic type.revious studies showed that Bik can be phosphorylated by protein kinase CKII (Casein Kinase 2). In order to mimic the post-translational phosphorylation process of Bik, Prof. Min-Chie Hung’s group generated a Bik mutant, BikDD (T33D/S35D). BikDD enhanced not only the binding affinity with Bcl-XL in vitro but also cancer cell killing ability in different cancer cell lines. Recently, they designed a pancreatic cancer specific promoter (CCKAR-VISA). By taking advantage of the specificity of this promoter, BikDD targeted pancreatic cancer cell in the pancreatic tumor-bearing mouse models. Pancreatic cancer cells were effectively killed and eradicated by BikDD. The significant anti-tumor results provide hopes for future cancer gene therapy in human clinical trials.he goal of this project is to determine the BikDD-BclXL complex structure. The structural information will provide solid evidence for Dr. Min-Chie Hung’s liposome-mediated BikDD cancer gene therapy. Both BikDD and Bcl-XL are membrane proteins. Their hydrophobic properties make themselves difficult to be purified and crystallized. We successfully over-expressed BikDD-BclXL complex in the E. coli system and tried different approaches to purify them from soluble or membrane fraction. In order to get a homogeneous sample for crystallization, we systematically screened different detergents, expressed different fragments, and tried bicelle crystallization methods. We utilized AUC and DLS technique to measure the protein homogeneity. After we obtained a homogeneous sample, we performed crystallization trials. n addition, we designed a platform for searching Bcl-XL inhibitors by using FRET. We hope this platform can help us to discover potential candidates for novel anti-cancer drug development.ACKNOWLEDGEMENTS...........................................IHINESE ABSTRACT..........................................IINGLISH ABSTRACT.........................................IIIABLE OF CONTENTS.........................................IVIST OF FIGURES...........................................VIIST OF TABLES...........................................VIIIST OF APPENDIX.........................................VIIIST OF ABBREVIATIONS................................. VIII hapter1. INTRODUCTION.....................................1.1 Roles of Bcl-2 Family in apoptosis.....................1.2 Homo- or Hetero- dimerization of Bcl-2 family proteins.2.3 Structural basis of Bcl-2 family proteins..............2.4 Anti-apoptotic proteins as targets for anticancer drug design.................................................3.5 Bik as a therapeutic agent for cancer gene therapy.....4.6 Goal of solving the BikDD - BclXL complex structure..5 hapter 2. MATERIALS and METHODS...........................6aterials .1 Reagents............................................6 .2 Instruments............................................7 Methods.3 Plasmid construction.................................8 .4 Site-directed mutagenesis.............................8 .5 Deletion of the loop region...........................8 .6 Protein over-expression..............................9 .7 Large-scale protein production......................9 .8 Protein purification...........................9 .9 Detergent tests......................................10.10 Crystallization................................10 .11 Analytical Ultracentrifuge (AUC)....................10.12 Dynamic light scattering (DLS)......................11.13 Bicelle crystallization.............................11 .14 FRET(Förster resonance energy transfer).............12hapter 3 RESULTS and DISCUSSION.........................13.1 BikDD and Bcl-XL can be co-expressed in the E. coli system...............................................13.2 Purification of the soluble BikDD-BclXL complex......13.3 Purification of BikDD-BCLXL complex from E.coli membrane.............................................14.4 Detergent exchange by using Gel-filtration column....16.5 Homogeneity analysis by using AUC and DLS............17.6 Deletion of the Bcl-XL loop region...................17 .7 Crystallization......................................18.8 Bicelle crystallization method.......................18.9 Co-expression of BikDD fragments with Bcl-XL△TM.....19hapter 4 CONCLUSION.....................................21EFERENCE................................................24IGURE...................................................26ABLE....................................................45PPENDIX.................................................48application/pdf5382003 bytesapplication/pdfen-USBcl-2 蛋白家族細胞凋亡膜蛋白BikDDBclXLBcl-2 familyApoptosisMembrane protein[SDGs]SDG3http://ntur.lib.ntu.edu.tw/bitstream/246246/178808/1/ntu-97-R95b46025-1.pdf