PMID: 9352091Jul 1, 1997

Synthesis of region-labelled proteins for NMR studies by in vitro translation of column-coupled mRNAs

Biochimie
M Y PavlovM Ehrenberg

Abstract

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.

References

Jul 1, 1979·Proceedings of the National Academy of Sciences of the United States of America·P C Jelenc, C G Kurland
Sep 11, 1979·Nucleic Acids Research·H Donis-Keller
Oct 1, 1975·FEBS Letters·N V BelitsinaS K Vasilenko
Oct 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·M H de Smit, J van Duin
Jan 1, 1989·Annual Review of Biochemistry·A Bax
Feb 1, 1971·Proceedings of the National Academy of Sciences of the United States of America·J S Dubnoff, U Maitra
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Citations

Nov 20, 1998·Current Opinion in Biotechnology·L JermutusA Plückthun
Feb 27, 1999·Current Opinion in Biotechnology·K J Rothschild, S Gite
Jun 4, 2008·Annual Review of Biochemistry·R Andrew MarshallJoseph D Puglisi

Related Concepts

Cell-Free System
In Vivo NMR Spectroscopy
Glucocorticoid Receptor
Poly(A) Tail
Protein Biosynthesis
Protein Engineering

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