Linked Life Data

11410295

Source:http://linkedlifedata.com/resource/pubmed/id/11410295

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2001-6-18
pubmed:abstractText
The rat tissue kallikrein rK9 is most abundant in the submandibular gland and the prostate. It has been successfully expressed in the Pichia pastoris yeast expression system. A full-length cDNA coding for the mature rK9 was fused in frame with yeast alpha-factor cDNA. The fusion protein was secreted into the medium with high yield without being processed by the yeast KEX2 signal peptidase. Mature rK9 was efficiently released from the fusion protein by trypsin and was purified to homogeneity by one-step affinity chromatography using soya bean trypsin inhibitor (SBTI) as affinity ligand. The identity of the recombinant enzyme was checked by N-terminal amino acid sequencing, Western blot analysis and kinetic studies. The dual trypsin- and chymotrypsin-like enzymatic specificity of rK9 was assessed by determining specificity constants (k(cat)/K(m)) for the hydrolysis of fluorogenic substrates, the peptide sequences of which were derived from proparathyroid hormone (pro-PTH) and from semenogelin-I. Our results confirmed the presence of an extended binding site in the rK9 active site. We also identified a far more sensitive substrate of this enzyme than those previously described, Abz-VKKRSARQ-EDDnp, which was hydrolysed with a catalytic efficiency k(cat)/K(m) of 420000 M(-1)s(-1). Finally, we showed that four of the five major proteins contained in secretions of rat seminal vesicles were rapidly degraded by recombinant rK9.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0006-3002
pubmed:author
pubmed:issnType
Print
pubmed:day
11
pubmed:volume
1547
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
387-96
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2001
pubmed:articleTitle
Purification and characterization of active recombinant rat kallikrein rK9.
pubmed:affiliation
Laboratory of Enzymology and Protein Chemistry, INSERM EMI-U 00-10, University François Rabelais, France.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't