Expression of a synthetic gene encoding canine milk lysozyme in Escherichia coli and characterization of the expressed protein

Takumi Koshiba, Tomohiro Hayashi, Ishido Miwako, Izumi Kumagai, Teikichi Ikura, Keiichi Kawano, Katsutoshi Nitta, Kunihiro Kuwajima

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27 Citations (Scopus)

Abstract

A high-expression plasmid of the canine milk lysozyme, which belongs to the family of calcium-binding lysozymes, was constructed in order to study its physico-chemical properties. Because the cDNA sequence of the protein has not yet been determined, a 400 base-pair gene encoding canine milk lysozyme was first designed on the basis of the known amino acid sequence. The gene was constructed by an enzymatic assembly of 21 chemically synthesized oligonucleotides and inserted into an Escherichia coli expression vector by stepwise ligation. The expression plasmid thus constructed was transformed into BL21(DE3)/pLysS cells. The gene product accumulated as inclusion bodies in an insoluble fraction. Recombinant canine milk lysozyme was obtained by purification and refolding of the product and showed the same characteristics in terms of bacteriolytic activity and far- and near-UV circular dichroism spectra as the authentic protein. The NMR spectra of refolded lysozyme were also characteristic of a native globular protein. It was concluded that recombinant canine milk lysozyme was folded into the correct native structure. Moreover, the thermal unfolding profiles of the refolded recombinant lysozyme showed a stable equilibrium intermediate, indicating that the molten globule state of this protein was extraordinarily stable. This expression system of canine milk lysozyme will enable biophysical and structural studies of this protein to be extended.

Original languageEnglish
Pages (from-to)429-435
Number of pages7
JournalProtein Engineering
Volume12
Issue number5
DOIs
Publication statusPublished - 1999

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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    Koshiba, T., Hayashi, T., Miwako, I., Kumagai, I., Ikura, T., Kawano, K., Nitta, K., & Kuwajima, K. (1999). Expression of a synthetic gene encoding canine milk lysozyme in Escherichia coli and characterization of the expressed protein. Protein Engineering, 12(5), 429-435. https://doi.org/10.1093/protein/12.5.429