Utilization of Track-etched Membrane Filter for a Crystallization Field and Single-crystal X-ray Diffraction Analysis of Proteins

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  • YAMADA Yohei
    National Institute of Technology, Anan College
  • SUZUTA Takahito
    Graduate School of Technology, Industrial and Social Sciences, Tokushima University
  • OKADA Eriko
    Graduate School of Technology, Industrial and Social Sciences, Tokushima University
  • TAKAYANAGI Toshio
    Graduate School of Technology, Industrial and Social Sciences, Tokushima University
  • SUZUKI Yoshihisa
    Graduate School of Technology, Industrial and Social Sciences, Tokushima University
  • MURAI Keiichiro
    Graduate School of Technology, Industrial and Social Sciences, Tokushima University
  • YABUTANI Tomoki
    Paper Industry Innovation Center, Ehime University

Bibliographic Information

Other Title
  • タンパク質結晶成長場及びX線回折実験用試料固定材としてのトラックエッチドメンブランフィルターの利用
  • タンパク質結晶成長場及びX線回析実験用試料固定材としてのトラックエッチドメンブランフィルターの利用
  • タンパクシツ ケッショウ セイチョウジョウ オヨビ Xセン カイセキ ジッケンヨウ シリョウ コテイザイ ト シテ ノ トラックエッチドメンブランフィルター ノ リヨウ

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Description

<p>Track-etched membrane filters (TEMF), which have highly homogeneous micropores, were applied to a protein crystal growth field and as a mounting material for synchrotron single-crystal X-ray diffraction analysis. In this study, lysozyme was utilized as the model protein for testing protein crystalline growth. TEMF with 10 or 30 μm pore size was treated with a lysozyme-crystalline growth solution, which contained lysozyme and NaCl as the crystalline growth assisting reagent in an acetate pH buffer solution, at 20°C. In order to evaluate the effect of TEMF on the lysozyme crystal growth, an optical microscope was employed to observe in situ the crystal growth by using an observation cell that provided a closed system to prevent the evaporation of the solvent in the crystalline solution. First, a crystalline core appeared within a pore, and then the crystal grew out of the pore. The analytical results of polarization microscopy and Raman spectroscopy suggested that the crystals on the TEMF were lysozyme crystals because the materials showed a polarization property and Raman scattering. The effect of TEMF on the crystal growth was evaluated and investigated in the presence and absence of TEMF using different concentrations of lysozyme and NaCl. The results showed that the presence of TEMF did not significantly affect the crystal growth. Further, the results of the solubility test of lysozyme crystals on TEMF showed that lysozyme crystals in the pores showed a higher stability than those outside the pores. In addition, the crystalline material in a single pore was analyzed by performing synchrotron single-crystal X-ray diffraction analysis (Spring-8 BL38B1, beam size 30 × 30 μm) using our handmade sample-mounting system consisting of a clip and magnet mount. Clear diffraction patterns were obtained from a single pore. The lattice constants from the diffraction pattern (a = b = 78.7 Å, c = 37.0 Å, α = β = γ = 90.0°) were quite similar to those of a tetragonal lysozyme crystal.</p>

Journal

  • BUNSEKI KAGAKU

    BUNSEKI KAGAKU 68 (9), 639-646, 2019-09-05

    The Japan Society for Analytical Chemistry

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