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Real-Time Single-Molecule Observation of Green Fluorescent Protein Synthesis by Immobilized Ribosomes

  • Ryo Iizuka
  • Takashi Funatsu
  • Sotaro UemuraEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 778)

Abstract

The dynamics of full protein synthesis and the co-translational folding processes are not fully understood. We have developed a novel method, using a combination of ribosome display and single-molecule techniques, for monitoring the synthesis, co-translational folding, and maturation of a complete polypeptide chain at the single-molecule level. This method enabled us to observe the appearance of green fluorescent protein fluorescence after de novo synthesis of the complete protein. Here, we provide the information necessary to reproduce this method, which will be valuable in revealing the dynamics of the co-translational folding and maturation of nascent polypeptides.

Key words

Ribosome display Translation Protein folding Green fluorescent protein SecM Translation arrest Single-molecule fluorescence imaging Total internal reflection fluorescence microscopy 

Notes

Acknowledgments

The authors thank Prof. Takuya Ueda and Dr. Yoshihiro Shimizu (The University of Tokyo) for the gift of plasmid pD-SecM-pURE1 and for helpful discussion, and Prof. Joseph D. Puglisi (Stanford University) for the gift of E. coli expressing C68 ribosomes.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Graduate School of Pharmaceutical SciencesUniversity of TokyoTokyoJapan
  2. 2.Omics Science Center, RIKEN Yokohama InstituteYokohamaJapan

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