Linked Life Data

10092497

Source:http://linkedlifedata.com/resource/pubmed/id/10092497

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
1999-5-3
pubmed:abstractText
The fbcB and fbcC genes encoding cytochromes b and c1 of the bc1 complex were extended with a segment to encode a polyhistidine tag linked to their C-terminal sequence allowing a one-step affinity purification of the complex. Constructions were made in vitro in a pUC-derived background using PCR amplification. The modified fbc operons were transferred to a pRK derivative plasmid, and this was used to transform the fbc- strain of Rhodobacter sphaeroides, BC17. The transformants showed normal rates of growth. Chromatophores prepared from these cells showed kinetics of turnover of the bc1 complex on flash activation which were essentially the same as those from wild-type strains, and analysis of the cytochrome complement and spectral and thermodynamic properties by redox potentiometry showed no marked difference from the wild type. Chromatophores were solubilized and mixed with Ni-NTA-Sepharose resin. A modification of the standard elution protocol in which histidine replaced imidazole increased the activity 20-fold. Imidazole modified the redox properties of heme c1, suggesting ligand displacement and inactivation when this reagent is used at high concentration. The purified enzyme contained all four subunits in an active dimeric complex. This construction provides a facile method for preparation of wild-type or mutant bc1 complex, for spectroscopy and structural studies.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
1046-5928
pubmed:author
pubmed:copyrightInfo
Copyright 1999 Academic Press.
pubmed:issnType
Print
pubmed:volume
15
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
370-80
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Expression and one-step purification of a fully active polyhistidine-tagged cytochrome bc1 complex from Rhodobacter sphaeroides.
pubmed:affiliation
Department of Biochemistry, University of Illinois, Urbana, Illinois, 61801, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.