Large-scale purification of ribosome-nascent chain complexes for biochemical and structural studies.
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.
Rutkowska
Anna
A
Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.
Beerbaum
Monika
M
Rajagopalan
Nandhakishore
N
Fiaux
Jocelyne
J
Schmieder
Peter
P
Kramer
Günter
G
Oschkinat
Hartmut
H
Bukau
Bernd
B
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification*; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism
D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D010455:Peptides; D011993:Recombinant Fusion Proteins