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

A high-expression plasmid of the canine milli lysozyme, which belongs to th e family of calcium-binding lysozymes, was constructed in order to study it s physico-chemical properties. Because the cDNA sequence of the protein has not yet been determined, a 400 base-pair gene encoding canine milk lysozym e was first designed on the basis of the known amino acid sequence. The gen e was constructed by an enzymatic assembly of 21 chemically synthesized oli gonucleotides and inserted into an Escherichia coli expression vector by st epwise ligation, The expression plasmid thus constructed was transformed in to 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 characterist ics in terms of bacteriolytic activity and far- and near-UV circular dichro ism spectra as the authentic protein. The NMR spectra of refolded lysozyme were also characteristic of a native globular protein. It was concluded tha t recombinant canine milk lysozyme was folded into the correct native struc ture, Moreover, the thermal unfolding profiles of the refolded recombinant lysozyme showed a stable equilibrium intermediate, indicating that the molt en globule state of this protein was extraordinarily stable. This expressio n system of canine milk lysozyme will enable biophysical and structural stu dies of this protein to be extended.