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Preparation of the Extracellular Domain of Recombinant Human Toll-like Receptor 6

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Abstract

Toll-like receptors (TLRs) mediate immune responses upon recognition of a variety of ligands. To further elucidate the function of TLRs, it is important to identify novel ligands and their action mechanisms including polymer assembly. In this study, we propose an efficient method for preparation of the extracellular domain of human Toll-like receptor 6 (TLR6ED) in Escherichia coli using the bubbling cultivation method. Our preparation method improved the level of expression of TLR6ED into a soluble fraction as compared with typical cultivation using a rotary shaker. Circular dichroism (CD) experiments confirmed the structural formation of TLR6ED with secondary structure contents similar to leucine-rich repeat (LRR) modules. In addition, we also provided a procedure for preparing this recombinant protein using Sf9 insect cells, which ensures preservation of some key posttranslational modifications often lacking in bacteria-expressed proteins. These materials would be useful for analyzing novel molecules that bind directly to TLR6, complex formations with other regulators including TLR2 and TLR4, and the functional effects of N-linked glycosylation.

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Abbreviations

TLR:

Toll-like receptor

LRR:

Leucine-rich repeat

PAMP:

Pathogen-associated molecular pattern

TLR6ED :

Extracellular domain of human TLR6

Sf9:

Spodoptera frugiperda 9

PCR:

Polymerase chain reaction

GST:

Glutathione S-transferase

LB:

Lysogeny-Broth

OD600 :

Optical density at 600 nm

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

SDS-PAGE:

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

CBB:

Coomassie Brilliant Blue

MOI:

Multiplicity of infection

PBS:

Phosphate buffered saline

CD:

Circular dichroism

DLS:

Dynamic light scattering

VLRB:

Variable lymphocyte receptor B

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Acknowledgements

This work was supported by a Grant-in-Aid for Scientific Research (S) (Grant Number 23228003) from the Japan Society for the Promotion of Science (JSPS) and the Platform for Drug Discovery, Informatics, and Structural Life Science of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

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Authors and Affiliations

  1. Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan

    Takuya Miyakawa, Yumiko Miyauchi & Masaru Tanokura

  2. Department of Health Promotion Sciences, Graduate School of Human Health Sciences, Tokyo Metropolitan University, 1-1, Minamiosawa, Hachioji City, Tokyo, 192-0397, Japan

    Ayane Kumazawa & Yoko Fuke

  3. Department of Life Sciences, Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, 562 Nanatsuka, Shobara, 727-0023, Japan

    Toshiro Noshita

  4. Center for Priority Areas, Tokyo Metropolitan University, 1-1, Minamiosawa, Hachioji City, Tokyo, 192-0397, Japan

    Masahiro Okada

Authors
  1. Takuya Miyakawa
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  2. Ayane Kumazawa
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  3. Yoko Fuke
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  4. Toshiro Noshita
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  5. Yumiko Miyauchi
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  6. Masahiro Okada
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  7. Masaru Tanokura
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Correspondence to Masaru Tanokura.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Miyakawa, T., Kumazawa, A., Fuke, Y. et al. Preparation of the Extracellular Domain of Recombinant Human Toll-like Receptor 6. Protein J 36, 28–35 (2017). https://doi.org/10.1007/s10930-016-9692-8

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  • DOI: https://doi.org/10.1007/s10930-016-9692-8

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