Linked Life Data

12356475

Source:http://linkedlifedata.com/resource/pubmed/id/12356475

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2002-10-1
pubmed:abstractText
The pET17 expression vector was used to express creatine kinase from the electric organ of Torpedo californica as inclusion bodies in Escherichia coli BL21(DE3) cells. The insoluble aggregate was dissolved in 8M urea and, following extraction with Triton X-100, the enzyme was refolded by dialysis against Tris buffer (pH 8.0) containing 0.2M NaCl. After two buffer changes, chromatography on Blue Sepharose was used as a final step in the purification procedure. Approximately 54mg active protein was recovered from a 1L culture and the refolded enzyme had a specific activity of 75U/mg. The molecular mass of the purified protein was consistent with that predicted from the amino acid sequence and the CD spectrum of the refolded enzyme was essentially identical to that of creatine kinase from human muscle (HMCK). The K(m) values of ATP and ADP were also similar to those of HMCK, while the K(m) values for both phosphocreatine and creatine were approximately 5-10-fold higher. The purification described here is in marked contrast with earlier attempts at purification of this isozyme where, in a process yielding less than 1mg/L culture, enzyme with a specific activity of ca. 5U/mg was obtained.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
1046-5928
pubmed:author
pubmed:issnType
Print
pubmed:volume
26
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
89-95
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2002
pubmed:articleTitle
Expression of Torpedo californica creatine kinase in Escherichia coli and purification from inclusion bodies.
pubmed:affiliation
College of Pharmacy, University of Michigan, 428 Church St., Ann Arbor, MI 48109-1065, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.