Linked Life Data

7560005

Source:http://linkedlifedata.com/resource/pubmed/id/7560005

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
1995-11-2
pubmed:abstractText
The eukaryotic organism Dictyostelium discoideum has become one of the organisms of choice for the overexpression of recombinant myosins and myosin fragments. Here, we describe a protocol that facilitates the screening of cells that have been transformed with myosin expression constructs and allows the rapid purification of recombinant myosins. Depletion of cellular ATP is used to recruit most of the endogenous and recombinant myosin into a rigor-like complex with actin. Following cell lysis the insoluble actomyosin complex is precipitated by centrifugation, washed, and Mg(2+)-ATP is added to extract the recombinant protein from the pellet. More than 90% of the protein in the resulting supernatant corresponds to actin, myosin, and the recombinant myosin fragments. Therefore, it is easy to detect any differences in expression level between individual myosin constructs on SDS-polyacrylamide gels. Additionally, the dependence of expression on external factors, such as cell density, can be readily determined. Furthermore, the presence of a band corresponding to the recombinant protein indicates that the overexpressed protein has at least some of the functional properties that are characteristic for a myosin motor. This rapid and selective extraction protocol can also be utilized to facilitate the purification of recombinant myosin motors on a preparative scale and has proved particularly useful in the purification of myosin head fragments, that are tagged with histidine residues, by Ni(2+)-chelate affinity chromatography.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0142-4319
pubmed:author
pubmed:issnType
Print
pubmed:volume
16
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
325-32
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed:year
1995
pubmed:articleTitle
Overexpression of myosin motor domains in Dictyostelium: screening of transformants and purification of the affinity tagged protein.
pubmed:affiliation
National Institute for Medical Research, London, UK.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't