Expression, purification and characterization of C-FADD.
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.
Chen
Yuan
Y
The State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu Center of Hepatobiliary Diseases, College of Life Sciences, Nanjing University, Nanjing 210093, China.
Ma
Dingyuan
D
Huang
Qi-Lai
QL
Zheng
Weijuan
W
Zhang
Jing
J
Shen
Yi
Y
Li
Jiahuang
J
Dong
Wei
W
Lu
Min
M
Wang
Jin
J
Hua
Zi-Chun
ZC
D000818:Animals; D017209:Apoptosis; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D053200:Fas-Associated Death Domain Protein / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D010446:Peptide Fragments / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics; D013329:Structure-Activity Relationship
C505534:Fadd protein, mouse; D053200:Fas-Associated Death Domain Protein; D010446:Peptide Fragments