SYNTHESIS OF REGION-LABELED PROTEINS FOR NMR-STUDIES BY IN-VITRO TRANSLATION OF COLUMN-COUPLED MESSENGER-RNAS

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 aci ds from the initiation codon of the mRNA and continues to the beginnin g of the region of interest. Here, the ribosomes pause while the tRNAs charged with unlabelled amino acids are replaced with tRNAs charged w ith isotope-labelled amino acids. Translation then proceeds through th e region of interest until the ribosomes pause at its end. At this poi nt aminoacyl tRNAs are changed again. Translation is resumed with unla belled amino acids and continues to the STOP codon of the mRNA, where the ribosomes pause. In the final step the complete, region labelled p rotein is eluted from the column in almost pure form. The method is de monstrated for small scale synthesis of the DNA binding domain (DBD) o f 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-label led.