PMID: 8589601Sep 1, 1995Paper

Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis

Journal of Biomolecular NMR
Takanori KigawaShigeyuki Yokoyama

Abstract

For the application of multidimensional NMR spectroscopy to larger proteins, it would be useful to perform selective labeling of one of the 20 amino acids. For some amino acids, however, amino acid metabolism drastically reduces the efficiency and selectivity of labeling in in vivo expression systems. In the present study, a cell-free protein synthesis system was optimized, so that highly efficient and selective stable isotope labeling of proteins can be achieved in the absence of amino acid metabolism. The productivity of the E. coli cell-free coupled transcription-translation system was first improved, by about fivefold, by using the T7 RNA polymerase for transcription and also by improving the translation conditions. Thus, about 0.1 mg protein per 1 ml reaction mixture was synthesized. Then, this improved cell-free system was used for Asp- or Ser-selective 15N-labeling of the human c-Ha-Ras protein. With a 15 ml cell-free reaction, using less than 1 mg of 15N-labeled amino acid, 1 mg of the Ras protein was obtained. 1H-15N HSQC experiments confirmed that the Ras protein was efficiently labeled with high selectivity. These results indicate that this cell-free protein synthesis system is useful for NMR studies.

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Citations

Jan 24, 2007·Journal of Biomolecular NMR·Takayoshi MatsudaTakanori Kigawa
Sep 12, 2007·Journal of Biomolecular NMR·Touraj Etezady-EsfarjaniKurt Wüthrich
Sep 3, 2008·Journal of Biomolecular NMR·Kit I TongToshiyuki Tanaka
Apr 29, 2009·Journal of Biomolecular NMR·Xinying JiaGottfried Otting
Aug 15, 2009·Journal of Biomolecular NMR·Solmaz SobhanifarVolker Dötsch
Nov 6, 2010·Journal of Biomolecular NMR·Jun YokoyamaTakanori Kigawa
Dec 12, 2012·Journal of Biomolecular NMR·Yoshikazu HattoriChojiro Kojima
Nov 30, 2012·Journal of Biomolecular NMR·Karin V Loscha, Gottfried Otting
Apr 23, 2003·Journal of Biochemical and Biophysical Methods·Didier BussoSung-Hou Kim
Jan 28, 2003·Current Opinion in Chemical Biology·Shigeyuki Yokoyama
Apr 8, 2010·Quarterly Reviews of Biophysics·Masatsune Kainosho, Peter Güntert
Mar 23, 2005·Nature Methods·Dmitriy A VinarovJohn L Markley
Jun 14, 2008·Proceedings of the National Academy of Sciences of the United States of America·Sina ReckelVolker Dötsch
Nov 8, 2001·Proceedings of the National Academy of Sciences of the United States of America·A ShimadaS Yokoyama
Oct 19, 2004·Genome Research·Joshua LabaerLeonardo Brizuela
Aug 1, 1996·European Journal of Biochemistry·D M KimS Yokoyama
Jan 20, 2004·European Journal of Biochemistry·Christian KlammtFrank Bernhard
Mar 31, 2010·Bioanalysis·Eugene Ciccimaro, Ian A Blair
Jun 14, 2014·Progress in Nuclear Magnetic Resonance Spectroscopy·Christoph GöblMichael Sattler
Aug 6, 2005·Biotechnology Progress·Kara A Calhoun, James R Swartz
Jun 22, 2005·Expert Review of Proteomics·Dmitriy A Vinarov, John L Markley
Sep 3, 2008·Expert Review of Proteomics·Joon ShinWeontae Lee
Mar 25, 2010·Expert Opinion on Drug Discovery·Robert Powers
Aug 12, 2009·Protein Expression and Purification·Masaaki AokiShigeyuki Yokoyama
Dec 13, 2006·Biotechnology and Bioengineering·Kurtis G KnappJames R Swartz
Apr 26, 2007·Protein Science : a Publication of the Protein Society·David F SavageRobert M Stroud
Aug 26, 2006·The FEBS Journal·Dmitriy A VinarovJohn L Markley
Jul 11, 2012·The FEBS Journal·Erich Michel, Kurt Wüthrich
Jan 1, 2014·Proteomics·Andreas OttoFrank Schmidt
Jul 11, 2006·Current Opinion in Biotechnology·Yaeta Endo, Tatsuya Sawasaki
Nov 13, 2007·Analytical Biochemistry·Michikazu TanioToshiyuki Kohno

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