Linked Life Data

15265740

Source:http://linkedlifedata.com/resource/pubmed/id/15265740

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2004-7-21
pubmed:abstractText
To extend the (strept)avidin-biotin technology for affinity purification of proteins, development of reusable biochips and immobilized enzyme bioreactors, selective immobilization of a protein of interest from a crude sample to a protein array without protein purification and many other possible applications, the (strept)avidin-biotin interaction is better when reversible. A gentle enzymatic method to introduce a biotin analog, desthiobiotin, in a site-specific manner to recombinant proteins carrying a biotinylation tag has been developed. The optimal condition for efficient in vitro desthiobiotinylation catalyzed by Escherichia coli biotin ligase (BirA) in 1-4h has been established by systematically varying the substrate concentrations, reaction time, and pH. Real desthiobiotinylation in the absence of any significant biotinylation using this enzymatic method was confirmed by mass spectrometric analysis of the desthiobiotinylated tag. This approach was applied to affinity purify desthiobiotinylated staphylokinase secreted by recombinant Bacillus subtilis to high purity and with good recovery using streptavidin-agarose. The matrix can be regenerated for reuse. This study represents the first successful application of E. coli BirA to incorporate biotin analog to recombinant proteins in a site-specific manner.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
0003-2697
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
331
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
340-8
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2004
pubmed:articleTitle
Development of an enzymatic method for site-specific incorporation of desthiobiotin to recombinant proteins in vitro.
pubmed:affiliation
Department of Biological Sciences, Division of Cellular, Molecular and Microbial Biology, University of Calgary, 2500 University Drive N.W., Calgary, Alberta, Canada T2N 1N4.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't