Linked Life Data

12842110

Source:http://linkedlifedata.com/resource/pubmed/id/12842110

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2003-7-4
pubmed:abstractText
Biochemical studies of eukaryotic proteins are often constrained by low availability of these typically large, multicomponent protein complexes in pure form. Escherichia coli is a commonly used host for large-scale protein production; however, its utility for eukaryotic protein production is limited because of problems associated with transcription, translation, and proper folding of proteins. Here we describe the development and testing of pLANT, a vector that addresses many of these problems simultaneously. The pLANT vector contains a T7 promoter-controlled expression unit, a p15A origin of replication, and genes for rare transfer RNAs and kanamycin resistance. Thus, the pLANT vector can be used in combination with the pET vector to coexpress multiple proteins in E. coli. Using this approach, we have successfully produced high-milligram quantities of two different Saccharomyces cerevisiae complexes in E. coli: the heterodimeric Msh2-Msh6 mismatch repair protein (248kDa) and the five-subunit replication factor C clamp loader (250 kDa). Quantitative analyses indicate that these proteins are fully active, affirming the utility of pLANT+pET-based production of eukaryotic proteins in E. coli for in vitro studies of their structure and function.
pubmed:grant
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
1
pubmed:volume
319
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
78-87
pubmed:dateRevised
2009-7-24
pubmed:meshHeading
pubmed:year
2003
pubmed:articleTitle
Overproduction and analysis of eukaryotic multiprotein complexes in Escherichia coli using a dual-vector strategy.
pubmed:affiliation
Rockefeller University and Howard Hughes Medical Institute, New York, NY 10021, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.