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Biotechnology Letters

, Volume 32, Issue 7, pp 897–902 | Cite as

N-terminally truncated GADD34 proteins are convenient translation enhancers in a human cell-derived in vitro protein synthesis system

  • Satoshi Mikami
  • Tominari Kobayashi
  • Kodai Machida
  • Mamiko Masutani
  • Shigeyuki Yokoyama
  • Hiroaki Imataka
Original Research Paper

Abstract

Human cell-derived in vitro protein synthesis systems are useful for the production of recombinant proteins. Productivity can be increased by supplementation with GADD34, a protein that is difficult to express in and purify from E. coli. Deletion of the N-terminal 120 or 240 amino acids of GADD34 improves recovery of this protein from E. coli without compromising its ability to boost protein synthesis in an in vitro protein synthesis system. The use of N-terminally truncated GADD34 proteins in place of full-length GADD34 should improve the utility of human cell-based cell-free protein synthesis systems.

Keywords

Cell-free system GADD34 In vitro translation Mammalian cells Protein synthesis 

Notes

Acknowledgements

This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (20200056) and a Grant-in-Aid for Scientific Research (C) (19580113) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan to H. I. and by the Targeted Proteins Research Program (TPRP) from MEXT.

References

  1. Brush MH, Shenolikar S (2008) Control of cellular GADD34 levels by the 26S proteasome. Mol Cell Biol 28:6989–7000CrossRefPubMedGoogle Scholar
  2. Brush MH, Weiser DC, Shenolikar S (2003) Growth arrest and DNA damage-inducible protein GADD34 targets protein phosphatase 1 alpha to the endoplasmic reticulum and promotes dephosphorylation of the alpha subunit of eukaryotic translation initiation factor 2. Mol Cell Biol 23:1292–1303CrossRefPubMedGoogle Scholar
  3. Carroll K, Elroy-Stein O, Moss B, Jagus R (1993) Recombinant vaccinia virus K3L gene product prevents activation of double-stranded RNA-dependent, initiation factor 2 alpha-specific protein kinase. J Biol Chem 268:12837–12842PubMedGoogle Scholar
  4. Mikami S, Kobayashi T, Yokoyama S, Imataka H (2006a) A hybridoma-based in vitro translation system that efficiently synthesizes glycoproteins. J Biotechnol 127:65–78CrossRefPubMedGoogle Scholar
  5. Mikami S, Masutani M, Sonenberg N, Yokoyama S, Imataka H (2006b) An efficient mammalian cell-free translation system supplemented with translation factors. Protein Expr Purif 46:348–357CrossRefPubMedGoogle Scholar
  6. Mikami S, Kobayashi T, Masutani M, Yokoyama S, Imataka H (2008) A human cell-derived in vitro coupled transcription/translation system optimized for production of recombinant proteins. Protein Expr Purif 62:190–198CrossRefPubMedGoogle Scholar
  7. Novoa I, Zeng H, Harding HP, Ron D (2001) Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2alpha. J Cell Biol 153:1011–1022CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Satoshi Mikami
    • 1
  • Tominari Kobayashi
    • 2
  • Kodai Machida
    • 2
  • Mamiko Masutani
    • 1
    • 2
  • Shigeyuki Yokoyama
    • 1
  • Hiroaki Imataka
    • 1
    • 2
  1. 1.RIKEN Systems and Structural Biology CenterYokohamaJapan
  2. 2.Department of Materials Science and Chemistry, Graduate School of EngineeringUniversity of HyogoHimejiJapan

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