Production and characterization of recombinant P1 adhesin essential for adhesion,gliding, and antigenic variation in the human pathogenic bacterium,Mycoplasma pneumoniae |
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Authors: | Tsuyoshi Kenri Yoshito Kawakita Hisashi Kudo U. Matsumoto Shigetarou Mori Yukio Furukawa Yuhei O. Tahara Keigo Shibayama Yuuki Hayashi Munehito Arai Makoto Miyata |
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Affiliation: | 1. Department of Bacteriology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo, 208-0011, Japan;2. Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka, 558-8585, Japan;3. Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo, 153-8902, Japan;4. Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan;5. The OCU Advanced Research Institute for Natural Science and Technology (OCARINA), Osaka City University, Osaka, Japan;6. Department of Physics, Graduate School of Science, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo, 153-8902, Japan |
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Abstract: | Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6?×?His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications. |
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Keywords: | Hydrodynamics Circular dichroism (CD) Electron microscopy Small-angle X-ray scattering (SAXS) Structure modeling Sialic acid receptor CD circular dichroism SAXS small-angle X-ray scattering IPTG PMSF phenylmethylsulfonyl fluoride EM electron microscopy |
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