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Large‐scale purification of ribosome‐nascent chain complexes for biochemical and structural studies
Author(s) -
Rutkowska Anna,
Beerbaum Monika,
Rajagopalan Nandhakishore,
Fiaux Jocelyne,
Schmieder Peter,
Kramer Günter,
Oschkinat Hartmut,
Bukau Bernd
Publication year - 2009
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2009.06.041
Subject(s) - ribosome , escherichia coli , folding (dsp implementation) , translation (biology) , chemistry , biophysics , protein folding , protein biosynthesis , chain (unit) , biochemistry , computational biology , microbiology and biotechnology , biology , messenger rna , rna , gene , physics , astronomy , electrical engineering , engineering
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.