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Suzuki T.,Clinical and Biotechnology Business Unit | Ezure T.,Clinical and Biotechnology Business Unit | Ando E.,Clinical and Biotechnology Business Unit | Nishimura O.,Osaka University | And 2 more authors.
Journal of Biotechnology | Year: 2010

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. © 2009 Elsevier B.V. All rights reserved.


Ezure T.,Clinical and Biotechnology Business Unit | Suzuki T.,Clinical and Biotechnology Business Unit | Shikata M.,Clinical and Biotechnology Business Unit | Ito M.,Okinawa National College of Technology | And 4 more authors.
Current Pharmaceutical Biotechnology | Year: 2010

Cell-free protein synthesis systems offer production of native proteins with high speed, even for the proteins that are toxic to cells. Among cell-free systems, the system derived from insect cells has the potential to carry out post-translational modifications that are specific to eukaryotic organisms, as occurs in the rabbit reticulocyte system. In this review, we describe development of this insect cell-free system and its applications. © 2010 Bentham Science Publishers Ltd.

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