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

Rutkowska, Anna; Beerbaum, Monika; Rajagopalan, Nandhakishore; Fiaux, Jocelyne; Schmieder, Peter; Kramer, Günter; Oschkinat, Hartmut; Bukau, Bernd

doi:10.1016/j.febslet.2009.06.041

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Volume 583, Issue 14 , Pages 2407-2413, 21 July 2009

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

Edited by Michael Ibba

Anna Rutkowska
- Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany
Monika Beerbaum
- Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany
Nandhakishore Rajagopalan
- Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany
Jocelyne Fiaux
- Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany
Peter Schmieder
- Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany
Günter Kramer
- Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany
Hartmut Oschkinat
- Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany
Bernd Bukau
- Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany
- Corresponding author.

Received 27 May 2009; received in revised form 17 June 2009; accepted 22 June 2009. published online 26 June 2009.

Abstract

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.

Keywords: Nascent chain, Protein folding, Ribosome, NMR