Purification and Crystallization of BikDD-BclXL complex
Date Issued
2008
Date
2008
Author(s)
Huang, Shao-Hsuan
Abstract
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.
Subjects
Bcl-2 family
Apoptosis
Membrane protein
SDGs
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