Protein Engineering, Vol. 12, No. 5, 429-435,
May 1999
© 1999 Oxford University Press
Expression of a synthetic gene encoding canine milk lysozyme in Escherichia coli and characterization of the expressed protein
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo 060–0810, 1 Department of Physics, School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113–0033 and 2 Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Aoba-ku, Sendai 980–8579, Japan
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.
Keywords: calcium-binding lysozyme/molten globule state/refolding/synthetic gene
3 Present address: Department of Biophysics, Faculty of Science, Kyoto University, Sakyo-ku, Kyoto 606–8501
4 Present address: Faculty of Pharmaceutical Science, Toyama Medical and Pharmaceutical University, Toyama, Toyama 930–0194, Japan
5 To whom correspondence should be addressed. E-mail: kuwajima{at}phys.s.u-tokyo.ac.jp
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  V. Casaite, S. Bruzyte, V. Bukauskas, A. Setkus, L. A. Morozova-Roche, and R. Meskys Expression and purification of active recombinant equine lysozyme in Escherichia coli Protein Eng. Des. Sel., November 1, 2009; 22(11): 649 - 654. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Koshiba, Y. Kobashigawa, M. Demura, and K. Nitta Energetics of three-state unfolding of a protein: canine milk lysozyme Protein Eng. Des. Sel., December 1, 2001; 14(12): 967 - 974. [Abstract] [Full Text] [PDF]
|
|