Title
Preparation of the Extracellular Domain of Recombinant Human Toll-like Receptor 6
Author
Takuya Miyakawa, Ayane Kumazawa, Yoko Fuke, Toshiro Noshita, Yumiko Miyauchi, Masahiro Okada, Masaru Tanokura
Year
2017
Journal
The Protein Journal
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.
Instrument
J-820
Keywords
Circular dichroism, Secondary structure, Biochemistry