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Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system

Paper ID Volume ID Publish Year Pages File Format Full-Text
24576 43527 2010 6 PDF Available
Title
Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system
Abstract

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.

Keywords
PTMs, posttranslational modifications; Ub, ubiquitin; UA, ubiquitin aldehyde; E3, E3 ubiquitin ligase; MS, mass spectrometry; Sf21, Spodoptera frugiperda 21; Me-Ub, methylated ubiquitin; MALDI-TOF MS, matrix assisted laser desorption/ionization time-of-fl
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Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 145, Issue 1, 1 January 2010, Pages 73–78
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us