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

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)683-690
Number of pages8
JournalProtein Engineering
Volume11
Issue number8
Publication statusPublished - Aug 1 1998

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Inclusion Bodies
Binding sites
Muramidase
Calcium
Enzymes
Binding Sites
Aspartic Acid
Temperature
Ions
Thioredoxins
Differential Scanning Calorimetry
Freezing
Chromatography
Gel Chromatography
Escherichia coli
Melting point
Differential scanning calorimetry
Gels

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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Calorimetric study of mutant human lysozymes with partially introduced Ca2+ binding sites and its efficient refolding system from inclusion bodies. / Koshiba, Takumi; Tsumoto, Kouhei; Masaki, Kazuo; Kawano, Keiichi; Nitta, Katsutoshi; Kumagai, Izumi.

In: Protein Engineering, Vol. 11, No. 8, 01.08.1998, p. 683-690.

Research output: Contribution to journalArticle

Koshiba, Takumi ; Tsumoto, Kouhei ; Masaki, Kazuo ; Kawano, Keiichi ; Nitta, Katsutoshi ; Kumagai, Izumi. / Calorimetric study of mutant human lysozymes with partially introduced Ca2+ binding sites and its efficient refolding system from inclusion bodies. In: Protein Engineering. 1998 ; Vol. 11, No. 8. pp. 683-690.
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