Linked Life Data

12830334

Source:http://linkedlifedata.com/resource/pubmed/id/12830334

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
7
pubmed:dateCreated
2003-10-30
pubmed:abstractText
Heterologous expression of domains of eukaryotic proteins is frequently associated with formation of inclusion bodies, consisting of aggregated mis-folded protein. This phenomenon has proved a significant barrier to the characterization of domains of eukaryotic ATP binding cassette (ABC) transporters. We hypothesized that the solubility of heterologously expressed nucleotide binding domains (NBDs) of ABC transporters is dependent on the definition of the domain boundaries. In this paper we have defined a core NBD, and tested the effect of extensions to and deletions of this core domain on protein expression. Of 10 NBDs constructed, only one was expressed as a soluble protein in Escherichia coli, with expression of the remaining NBDs being associated with inclusion body formation. The soluble NBD protein we have obtained corresponds to residues 386-632 of P-glycoprotein and represents an optimally defined domain. The NBD has been isolated and purified to 95% homogeneity by a two-step purification protocol, involving affinity chromatography and gel filtration. Although showing no detectable ATP hydrolysis, the protein retains specific ATP binding and has a secondary structure compatible with X-ray crystallographic data on bacterial NBDs. We have interpreted our results in terms of homology models, which suggest that the N-terminal NBD of P-glycoprotein can be produced as a stable, correctly folded, isolate domain with judicious design of the expression construct.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
0175-7571
pubmed:author
pubmed:issnType
Print
pubmed:volume
32
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
644-54
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2003
pubmed:articleTitle
Definition of the domain boundaries is critical to the expression of the nucleotide-binding domains of P-glycoprotein.
pubmed:affiliation
School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK. ian.kerr@nottingham.ac.uk
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't