Expressed protein ligation, a novel method for studying protein-protein interactions in transcription.

The Journal of Biological Chemistry
K Severinov, Tom W Muir

Abstract

Expressed protein ligation is a novel protein semi-synthesis method that permits the in vitro ligation of a chemically synthesized C-terminal segment of a protein to a recombinant N-terminal segment fused through its C terminus to an intein protein splicing element. In principle, the practical convenience of this method, combined with the expanded opportunities in protein engineering that it provides, makes it well suited for probing the molecular basis of complex processes such as transcription. Here we describe the successful application of expressed protein ligation to the approximately 600 amino acid sigma70 subunit of Escherichia coli RNA polymerase. The resulting semi-synthetic sigma70 constructs are shown to be fully functional and have been used to map the binding region of the bacteriophage T4 anti-sigma protein, AsiA, to within amino acids 567-600 of sigma70. The success of these semi-synthesis studies sets the stage for the future generation of semi-synthetic sigma70 molecules in which unnatural amino acids and biophysical probes are site-specifically incorporated in the RNA polymerase complex.

References

May 1, 1992·Bioconjugate Chemistry·H F GaertnerR E Offord
Sep 1, 1992·International Journal of Peptide and Protein Research·M SchnölzerS B Kent
Jun 1, 1992·Journal of Bacteriology·M LonettoC A Gross
Apr 20, 1989·Journal of Molecular Biology·T GardellaM M Susskind
Aug 1, 1995·Current Opinion in Biotechnology·C J Wallace
Nov 4, 1994·Science·P E DawsonS B Kent
Jan 14, 1994·Journal of Molecular Biology·A KumarA Ishihama
Jul 15, 1996·Structure·W L HubbellM A Lietzow
Nov 15, 1996·Journal of Molecular Biology·E SeverinovaS A Darst
Apr 10, 1997·Nature·M Ptashne, A Gann
Feb 12, 1998·Proceedings of the National Academy of Sciences of the United States of America·K Severinov, S A Darst
Apr 29, 1998·The Journal of Peptide Research : Official Journal of the American Peptide Society·J A CamareroT W Muir
Jun 17, 1998·Proceedings of the National Academy of Sciences of the United States of America·T W MuirP A Cole

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Citations

Feb 24, 2000·Biopolymers·J P TamZ Miao
Jan 5, 2002·Biopolymers·J P TamK D Eom
Aug 18, 2009·Cellular and Molecular Life Sciences : CMLS·Luis Berrade, Julio A Camarero
May 8, 2010·Applied Microbiology and Biotechnology·Skander Elleuche, Stefanie Pöggeler
Aug 18, 2009·Amino Acids·Jeffrey AustinJulio A Camarero
Aug 20, 2009·Journal of Biomolecular NMR·Lenka SkrisovskaFrédéric H-T Allain
Dec 29, 2010·Journal of Biomolecular NMR·Mary Anne RefaeiPearl Tsang
Dec 9, 2003·Journal of Molecular Biology·Marina ZakharovaKonstantin Severinov
Sep 7, 2000·Current Opinion in Biotechnology·F B Perler, E Adam
Sep 27, 2002·Current Opinion in Biotechnology·Roseanne M Hofmann, Tom W Muir
Aug 28, 1998·Current Opinion in Biotechnology·J Wilken, S B Kent
Oct 2, 2001·Current Opinion in Chemical Biology·T Ozawa, Y Umezawa
Dec 22, 1999·Current Opinion in Chemical Biology·G G Kochendoerfer, S B Kent
Jun 13, 2012·Journal of the American Chemical Society·Jingjing J LingBradley L Pentelute
Jun 16, 2012·The Journal of Biological Chemistry·James P Tam, Clarence T T Wong
Dec 18, 2001·The EMBO Journal·L J LambertM H Werner
Feb 22, 2011·Protein Engineering, Design & Selection : PEDS·G Elif KaragözStefan G D Rüdiger
Jan 13, 2000·Protein Science : a Publication of the Protein Society·B OdaertG Lippens
Aug 10, 2007·Molekuliarnaia biologiia·K V Severinov
Mar 11, 2003·Annual Review of Biochemistry·Tom W Muir
Oct 30, 2010·Virology Journal·Deborah M Hinton
Oct 11, 2001·Biological Chemistry·M VölkertH Waldmann
Jan 20, 1999·Proceedings of the National Academy of Sciences of the United States of America·H Yu
Jan 20, 1999·Proceedings of the National Academy of Sciences of the United States of America·R XuT W Muir
Jun 27, 2002·Proceedings of the National Academy of Sciences of the United States of America·Julio A CamareroTom W Muir
Dec 2, 2015·Proceedings of the National Academy of Sciences of the United States of America·Rina RosenzweigLewis E Kay
Oct 21, 2014·Chemical Reviews·Manuel M Müller, Tom W Muir
May 20, 2003·Molecular Microbiology·Simon L DoveAnn Hochschild
Mar 31, 2004·The Journal of Peptide Research : Official Journal of the American Peptide Society·C T PoolJ P Tam
Oct 1, 2009·Journal of Peptide Science : an Official Publication of the European Peptide Society·Stevenson Flemer
May 8, 2004·Molecular Microbiology·Gilbert OrsiniAnnie Kolb
Jul 17, 2008·Protein Science : a Publication of the Protein Society·Nikhil SinglaDimitar B Nikolov
Dec 20, 2005·The EMBO Journal·Francesca VitaliFrédéric H-T Allain

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