Synthesis of region-labelled proteins for NMR studies by in vitro translation of column-coupled mRNAs.
A method to synthesise region-labelled proteins for structural studies with NMR is suggested. The technique is based on in vitro translation of matrix-coupled mRNAs. Translation starts with unlabelled amino acids from the initiation codon of the mRNA and continues to the beginning of the region of interest. Here, the ribosomes pause while the tRNAs charged with unlabelled amino acids are replaced with tRNAs charged with isotope-labelled amino acids. Translation then proceeds through the region of interest until the ribosomes pause at its end. At this point aminoacyl-tRNAs are changed again. Translation is resumed with unlabelled amino acids and continues to the STOP codon of the mRNA, where the ribosomes pause. In the final step the complete, region-labelled protein is eluted from the column in almost pure form. The method is demonstrated for small scale synthesis of the DNA binding domain (DBD) of the glucocorticoid receptor (GR), where the DNA-recognising helix is labelled but the rest of DBD is unlabelled. The new technique can be generalised to allow a desired region in a protein to be isotope-labelled.
Pavlov
M Y
MY
Department of Molecular Biology, BMC, Uppsala, Sweden.
Freistroffer
D V
DV
Ehrenberg
M
M
D000595:Amino Acid Sequence; D002474:Cell-Free System; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry*; D011965:Receptors, Glucocorticoid / Q000737:chemistry / Q000235:genetics
D011506:Proteins; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid