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


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. Study holds ProTherm entries: 13147, 13148, 13149, 13150 Extra Details: Holo-lysozyme in the presence of 10 mM CaCl2 calcium-binding lysozyme; molten globule state; refolding; synthetic gene

Submission Details

ID: j7PV7tDa

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:44 p.m.

Version: 1

Publication Details
Koshiba T;Hayashi T;Miwako I;Kumagai I;Ikura T;Kawano K;Nitta K;Kuwajima K,Protein Eng. (1999) Expression of a synthetic gene encoding canine milk lysozyme in Escherichia coli and characterization of the expressed protein. PMID:10360984
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1I56 2002-02-27 SOLUTION STRUCTURE OF CA2+-BOUND STATE OF CANINE MILK LYSOZYME
1QQY 2000-06-09 1.85 X-RAY CRYSTAL STRUCTURE ANALYSIS OF CANINE MILK LYSOZYME (APO-TYPE)
1EL1 2001-03-13 1.9 X-RAY CRYSTAL STRUCTURE ANALYSIS OF CANINE MILK LYSOZYME (HOLO-TYPE)
2CWI 2006-06-20 1.94 X-ray crystal structure analysis of recombinant wild-type canine milk lysozyme (apo-type)
2Z2E 2007-11-27 2.01 Crystal Structure of Canine Milk Lysozyme Stabilized against Non-enzymatic Deamidation

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Lysozyme C, milk isozyme P81708 LYSC1_CANLF