Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.
Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.
Suzuki
Takashi
T
Life Science Business Department, Shimadzu Corporation, Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto, Japan. t-suzuki@shimadzu.co.jp
Ezure
Toru
T
Ando
Eiji
E
Nishimura
Osamu
O
Utsumi
Toshihiko
T
Tsunasawa
Susumu
S
D000818:Animals; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002474:Cell-Free System / Q000378:metabolism*; D025821:Polyubiquitin / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination
D011506:Proteins; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D025821:Polyubiquitin; D020169:Caspases