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Cell-free synthesis of human toll-like receptor 9 (TLR9): Optimization of synthesis conditions and functional analysis

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  • Srujan Kumar Dondapati
  • Georg Pietruschka
  • Lena Thoring
  • Doreen A Wüstenhagen
  • Stefan Kubick

Abstract

The Toll-like receptor family belongs to the group of pathogen recognition receptors which is responsible for the discrimination of self and non-self pathogen-associated molecular patterns (PAMP’s). Toll-like receptors play an important role in the innate immunity and defects in protein expression or polymorphism is linked to various diseases such as Systemic Lupus Erythematosus (SLE). The elucidation of the underlying mechanism is crucial for future treatment and therapeutics of toll-like receptor linked diseases. Herein, we report the cell-free synthesis of human Toll-like receptor 9 (hTLR9) using CHO lysate and the continuous exchange cell-free (CECF) synthesis platform. The functionality of this protein was demonstrated by an ELISA binding assay using the ectodomain of TLR9 (TLR9-ECD).

Suggested Citation

  • Srujan Kumar Dondapati & Georg Pietruschka & Lena Thoring & Doreen A Wüstenhagen & Stefan Kubick, 2019. "Cell-free synthesis of human toll-like receptor 9 (TLR9): Optimization of synthesis conditions and functional analysis," PLOS ONE, Public Library of Science, vol. 14(4), pages 1-16, April.
    • Handle: RePEc:plo:pone00:0215897
    DOI: 10.1371/journal.pone.0215897
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    1. Umeharu Ohto & Takuma Shibata & Hiromi Tanji & Hanako Ishida & Elena Krayukhina & Susumu Uchiyama & Kensuke Miyake & Toshiyuki Shimizu, 2015. "Structural basis of CpG and inhibitory DNA recognition by Toll-like receptor 9," Nature, Nature, vol. 520(7549), pages 702-705, April.
    2. Sarah E. Ewald & Bettina L. Lee & Laura Lau & Katherine E. Wickliffe & Guo-Ping Shi & Harold A. Chapman & Gregory M. Barton, 2008. "The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor," Nature, Nature, vol. 456(7222), pages 658-662, December.
    3. Hiroaki Hemmi & Osamu Takeuchi & Taro Kawai & Tsuneyasu Kaisho & Shintaro Sato & Hideki Sanjo & Makoto Matsumoto & Katsuaki Hoshino & Hermann Wagner & Kiyoshi Takeda & Shizuo Akira, 2000. "A Toll-like receptor recognizes bacterial DNA," Nature, Nature, vol. 408(6813), pages 740-745, December.
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