Linked Life Data

10360984

Source:http://linkedlifedata.com/resource/pubmed/id/10360984

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
5
pubmed:dateCreated
1999-7-21
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0269-2139
pubmed:author
pubmed:issnType
Print
pubmed:volume
12
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
429-35
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Expression of a synthetic gene encoding canine milk lysozyme in Escherichia coli and characterization of the expressed protein.
pubmed:affiliation
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo 060-0810, Japan.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't