Screening and characterization of a novel fibrinolytic metalloprotease from a metagenomic library.
A metagenomic library was constructed using total genomic DNA extracted from the mud in the west coast of Korea and was used together with a fosmid vector, pCC1FOS in order to uncover novel gene sources. One clone from approximately 30,000 recombinant Escherichia coli clones was identified that showed proteolytic activity. The gene for the proteolytic enzyme was subcloned into pUC19 and sequenced, and a database search for homologies revealed it to be a zinc-dependent metalloprotease. The cloned gene included the intact coding gene for a novel metalloproteinase and its own promoter. It comprised an open reading frame of 1,080 base pairs, which encodes a protein of 39,490 Da consisting of 359 amino acid residues. A His-Glu-X-X-His sequence, which is a conserved sequence in the active site of zinc-dependent metalloproteases, was found in the deduced amino acid sequence of the gene, suggesting that the enzyme is a zinc-dependent metalloprotease. The purified enzyme showed optimal activity at 50 degrees C for 1 h and pH 7.0. The enzyme activity was inhibited by metal-chelating reagents, such as EDTA, EGTA and 1,10-phenanthroline. The enzyme hydrolyzed azocasein as well as fibrin. Thus, the enzyme could be useful as a therapeutic agent to treat thrombosis.
Lee
Dong-Geun
DG
Department of Pharmaceutical Engineering, College of Medical Life Science, Silla University, Busan, Korea.
Jeon
Jeong Ho
JH
Jang
Min Kyung
MK
Kim
Nam Young
NY
Lee
Jong Hyun
JH
Lee
Jung-Hyun
JH
Kim
Sang-Jin
SJ
Kim
Gun-Do
GD
Lee
Sang-Hyeon
SH
D001426:Bacterial Proteins / Q000235:genetics; D002874:Chromosome Mapping / Q000379:methods*; D015195:Drug Design; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000737:chemistry*; D019015:Geologic Sediments / Q000382:microbiology*; D045726:Metalloproteases / Q000737:chemistry* / Q000235:genetics*; D019151:Peptide Library; D020543:Proteome / Q000235:genetics*
D001426:Bacterial Proteins; D005343:Fibrinolytic Agents; D019151:Peptide Library; D020543:Proteome; D045726:Metalloproteases