Calorimetric study of mutant human lysozymes with partially introduced Ca2+ binding sites and its efficient refolding system from inclusion bodies

Takumi Koshiba, Kouhei Tsumoto, Kazuo Masaki, Keiichi Kawano, Katsutoshi Nitta, Izumi Kumagai

研究成果: ジャーナルへの寄稿記事

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During the process of evolution, ancestral lysozymes evolved into calcium-binding lysozymes by acquiring three critical aspartate residues at positions 86, 91 and 92. To investigate the process of the acquisition of calcium-binding ability, two of the aspartates were partially introduced into human lysozyme at positions 86, 91 and 92. These mutants (HLQ86D, HLA92D and HLQ86D/D91Q/A92D), having two critical aspartates in calcium-binding sites, were expressed in Escherichia coli as non-active inclusion bodies. For the preparation of lysozyme samples, a refolding system using thioredoxin was established. This system allowed for effective refolding of wild-type and mutant lysozymes, and 100% of activity was recovered within 4 days. The calcium ion dependence of the melting temperature (T(m)) of wild-type and mutant lysozymes was investigated by differential scanning calorimetry at pH 4.5. The T(m) values of wild-type, HLQ86D and HLA92D mutants were not dependent on calcium ion concentration. However, the T(m) of HLQ86D/ D91Q/A92D was 4°higher in the presence of 50 mM CaCl2 than in its absence, and the calcium-binding constant of this mutant was estimated to be 2.25(± 0.25) x 102 M-1 at pH 4.5. Moreover, the calcium-binding ability of this mutant was confirmed by the result using Sephadex G-25 gel chromatography. These results indicate that it is indispensable to have at least two aspartates at positions 86 and 92 for acquisition of calcium-binding ability. The process of the acquisition of calcium-binding site during evolution of calcium-binding lysozyme is discussed.

元の言語英語
ページ(範囲)683-690
ページ数8
ジャーナルProtein Engineering
11
発行部数8
出版物ステータス出版済み - 8 1 1998

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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