Large-scale purification of ribosome-nascent chain complexes for biochemical and structural studies |
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Authors: | Anna Rutkowska Nandhakishore Rajagopalan Peter Schmieder Hartmut Oschkinat |
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Affiliation: | a Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany b Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany |
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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. |
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Keywords: | Nascent chain Protein folding Ribosome NMR |
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