Large-scale purification of ribosome-nascent chain complexes for biochemical and structural studies

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• 作者：Anna Rutkowska ; Monika Beerbaum ; N ; hakishore Rajagopalan ; Jocelyne Fiaux ; Peter Schmieder ; Gü ; nter Kramer ; Hartmut Oschkinat ; Bernd Bukau
• 关键词：Nascent chain ; Protein folding ; Ribosome ; NMR
• 刊名：FEBS Letters
• 出版年：2009
• 出版时间：21 July 2009
• 年：2009
• 卷：583
• 期：14
• 页码：2407-2413
• 全文大小：1077 K

文摘

Here we present a method to purify large amounts of highly pure and stably arrested ribosome-nascent chain complexes (RNCs) from Escherichia coli cells. It relies on the combined use of translation-arrest sequences to generate nascent polypeptides of specified length and subsequent tag purification of the RNCs. Moreover, we adapted this method for the in vivo production of RNCs with specific isotope labeling of the nascent chains for nuclear magnetic resonance (NMR) studies. This method opens therefore possibilities for a wide range of biochemical and structural studies exploring conformations of nascent chains during the early steps of protein folding and targeting.