Expression, Purification and Characterization of C-FADD

• Published in: Cellular & molecular immunology Vol. 6; no. 4; pp. 295 - 301
• Main Authors: Chen, Yuan, Ma, Dingyuan, Huang, Qi-Lai, Zheng, Weijuan, Zhang, Jing, Shen, Yi, Li, Jiahuang, Dong, Wei, Lu, Min, Wang, Jin, Hua, Zi-Chun
• Format: Journal Article
• Language: English
• Published: China Nature Publishing Group 01.08.2009; Changzhou High-Tech Research Institute of Nanjing University,Changzhou 213164,China; The State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu Center of Hepatobiliary Diseases,College of Life Sciences,Nanjing University,Nanjing 210093,China%The State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu Center of Hepatobiliary Diseases,College of Life Sciences,Nanjing University,Nanjing 210093,China
• Subjects: Adaptor proteins; Affinity chromatography; Animals; Apoptosis; Cell cycle; Cell survival; Chromatography, Affinity; Circular Dichroism; Cloning, Molecular; Crystallization; Escherichia coli; Escherichia coli - genetics; FADD protein; Fas-Associated Death Domain Protein - genetics; Fas-Associated Death Domain Protein - isolation & purification; Fas-Associated Death Domain Protein - metabolism; Mice; N-Terminus; Peptide Fragments - genetics; Peptide Fragments - isolation & purification; Peptide Fragments - metabolism; Protein Binding; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary - genetics; Structure-Activity Relationship; Structure-function relationships; C-FADD; expression; monomer; purification
• ISSN: 1672-7681; 2042-0226
• DOI: 10.1038/cmi.2009.39

FADD is an important proapoptotic adaptor in death receptor-induced apoptosis. Recently, FADD has been found to participate in a variety of non-apoptotic processes, such as development, cell cycle progression and survival. Its non-apoptotic activities were regulated by the phosphorylated status of the serine residue located at the C-terminal region, a domain distinct from the proapoptotic function related DED and DD domains. However, due to the difficulties in expression and crystallization of natural FADD, by far the molecular structures of all FADD variants did not contain the C-terminal region. To elucidate the structure-function relationship of C-terminal region, we need to obtain an FADD variant that containing C-terminal region. In this study, mouse FADD (80-205) containing DD domain and C-terminal region, designated as C-FADD, was expressed in E. coli with His-tag at the N-terminus and purified by Ni2+ affinity chromatography. The purified protein existed as a homogenous monomer in glutaraldehyde cross-linking analysis and exhibited a typical alpha-helix spectrum in CD (circular dichroism) assay. In vitro His-tag pull-down assay demonstrated that the purified C-FADD possessed the CK Ialpha-binding activity which was important for its non-apoptotic function.