Linked Life Data

10648173

Source:http://linkedlifedata.com/resource/pubmed/id/10648173

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2000-3-9
pubmed:abstractText
The cytoplasmic tyrosine kinase p72syk (Syk) plays an essential role in signaling via a variety of immune and nonimmune cell receptors. Syk is activated in response to the engagement of the appropriate cell surface receptors and can phosphorylate downstream targets and recruit additional SH2-domain-containing proteins. In order to study the characteristics of Syk in vitro, we have overexpressed untagged, full-length human Syk in a recombinant baculovirus expression system. The enzyme was purified to 95% purity using a novel two-step affinity chromatography process using reactive yellow and phosphotyrosine columns. Yields of 3-10 mg purified Syk were obtained from 1 liter of infected insect cells. Western blotting, internal protein sequencing, and the specific tyrosine phosphorylation of a Syk peptide substrate indicated authenticity of the purified protein. The enzymatic properties of Syk were in good agreement with published data for the human enzyme, as the apparent K(m) of Syk for ATP was 10 microM and the peptide substrate was 3 microM. The recombinant protein also showed similar biochemical characteristics to the native protein isolated from B-cells such as autophosphorylation. Proteolytic cleavage of purified recombinant Syk was used to generate the kinase domain by micro-calpain. We therefore describe an efficient expression system and purification methodology to produce biologically active human Syk.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
1046-5928
pubmed:author
pubmed:copyrightInfo
Copyright 2000 Academic Press.
pubmed:issnType
Print
pubmed:volume
18
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
86-94
pubmed:dateRevised
2011-11-2
pubmed:meshHeading
pubmed:year
2000
pubmed:articleTitle
Purification and characterization of human Syk produced using a baculovirus expression system.
pubmed:affiliation
Respiratory Disease Therapeutic Area, Novartis Horsham Research Centre, Wimblehurst Road, Horsham, West Sussex, RH12 5AB, United Kingdom. Darren.Baldock@pharma.novartis.com
pubmed:publicationType
Journal Article