Preparation of the Extracellular Domain of Recombinant Human Toll-like Receptor 6.
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 TLR6EDinto a soluble fraction as compared with typical cultivation using a rotary shaker. Circular dichroism (CD) experiments confirmed the structural formation of TLR6EDwith 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.
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
T
Miyakawa
Department of Health Promotion Sciences, Graduate School of Human Health Sciences, Tokyo Metropolitan University, 1-1, Minamiosawa, Hachioji City, Tokyo, 192-0397, Japan.
Ayane
A
Kumazawa
Department of Health Promotion Sciences, Graduate School of Human Health Sciences, Tokyo Metropolitan University, 1-1, Minamiosawa, Hachioji City, Tokyo, 192-0397, Japan.
Yoko
Y
Fuke
Department of Life Sciences, Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, 562 Nanatsuka, Shobara, 727-0023, Japan.
Toshiro
T
Noshita
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.
Yumiko
Y
Miyauchi
Center for Priority Areas, Tokyo Metropolitan University, 1-1, Minamiosawa, Hachioji City, Tokyo, 192-0397, Japan.
Masahiro
M
Okada
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. amtanok@mail.ecc.u-tokyo.ac.jp.
Masaru
0000-0001-5072-2480
M
Tanokura
D000818:Animals; D002942:Circular Dichroism; D004926:Escherichia coli; D006801:Humans; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D061987:Sf9 Cells; D018411:Spodoptera; D051216:Toll-Like Receptor 6 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
D011994:Recombinant Proteins; C495368:TLR6 protein, human; D051216:Toll-Like Receptor 6
Bubbling cultivation; Circular dichroism; Dynamic light scattering; Toll-like receptor