Linked Life Data

9387243

Source:http://linkedlifedata.com/resource/pubmed/id/9387243

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:dateCreated
1998-2-10
pubmed:abstractText
Detailed molecular analysis of the Rickettsia prowazekii ATP/ADP translocase, an obligate exchange transport system that is specific for ATP and ADP, has been extremely difficult due to limited quantities of material available from these obligate intracytoplasmic bacteria and by the toxicity and poor expression in recombinant Escherichia coli expression systems. In this study, a stable and controllable system for the increased expression of the rickettsial ATP/ADP translocase was developed in E. coli where the expression of translocase from the bacteriophage T7 promoter in the pET11a vector led to a 26-fold increase in ATP transport activity and a 34-fold increase in translocase protein as compared to the expression with the native rickettsial promoter in E. coli. When compared to R. prowazekii, ATP transport activity was increased sixfold and membrane translocase was increased threefold. Approximately 24% of the translocase protein produced was localized in an inclusion body fraction. This expression system was then used to determine whether the two cysteine residues in the ATP/ADP translocase were essential for activity or expression. The translocase was modified by oligonucleotide-directed site-specific mutagenesis such that the two cysteines were converted to alanines. The ATP transport properties and ATP/ADP translocase production kinetics, translocase protein concentration and subcellular localization were indistinguishable in the wild-type and mutant strains, proving that cysteines play no functional role in the R. prowazekii ATP/ADP translocase and providing a system suitable for cysteine-scanning mutagenesis.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
1350-0872
pubmed:author
pubmed:issnType
Print
pubmed:volume
143 ( Pt 11)
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
3661-9
pubmed:dateRevised
2008-11-21
pubmed:meshHeading
pubmed:year
1997
pubmed:articleTitle
Increased and controlled expression of the Rickettsia prowazekii ATP/ADP translocase and analysis of cysteine-less mutant translocase.
pubmed:affiliation
Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile 36688, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.