Linked Life Data

18191412

Source:http://linkedlifedata.com/resource/pubmed/id/18191412

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2008-2-27
pubmed:abstractText
N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, initiates excision repair of several N-alkylpurine adducts, deaminated and lipid peroxidation-induced purine adducts. MPG from human and mouse has previously been cloned and expressed. However, due to the poor expression level in Escherichia coli (E. coli) and multi-step purification process of full-length MPG, most successful attempts have been limited by extremely poor yield and stability. Here, we have optimized the codons within the first five residues of human MPG (hMPG) to the best used codons for E. coli and expressed full-length hMPG in large amounts. This high expression level in conjunction with a strikingly high isoelectric point (9.65) of hMPG, in fact, helped purify the enzyme in a single step. A previously well-characterized monoclonal antibody having an epitope in the N-terminal tail could detect this codon-optimized hMPG protein. Surface plasmon resonance studies showed an equilibrium binding constant (K(D)) of 0.25 nM. Steady-state enzyme kinetics showed an apparent K(m) of 5.3 nM and k(cat) of 0.2 min(-1) of MPG for the hypoxanthine (Hx) cleavage reaction. Moreover, hMPG had an optimal activity at pH 7.5 and 100mM KCl. Unlike the previous reports by others, this newly purified full-length hMPG is appreciably stable at high temperature, such as 50 degrees C. Thus, this study indicates that this improved expression and purification system will facilitate large scale production and purification of a stable human MPG protein for further biochemical, biophysical and structure-function analysis.
pubmed:grant
pubmed:commentsCorrections
http://linkedlifedata.com/resource/pubmed/commentcorrection/18191412-11106395, http://linkedlifedata.com/resource/pubmed/commentcorrection/18191412-12016206, http://linkedlifedata.com/resource/pubmed/commentcorrection/18191412-12144783, http://linkedlifedata.com/resource/pubmed/commentcorrection/18191412-16901897, http://linkedlifedata.com/resource/pubmed/commentcorrection/18191412-17716976, http://linkedlifedata.com/resource/pubmed/commentcorrection/18191412-4000169, http://linkedlifedata.com/resource/pubmed/commentcorrection/18191412-6371006, http://linkedlifedata.com/resource/pubmed/commentcorrection/18191412-7999773, http://linkedlifedata.com/resource/pubmed/commentcorrection/18191412-8016081, http://linkedlifedata.com/resource/pubmed/commentcorrection/18191412-8034633, http://linkedlifedata.com/resource/pubmed/commentcorrection/18191412-8110764, http://linkedlifedata.com/resource/pubmed/commentcorrection/18191412-8284199, http://linkedlifedata.com/resource/pubmed/commentcorrection/18191412-8469282, http://linkedlifedata.com/resource/pubmed/commentcorrection/18191412-8706136, http://linkedlifedata.com/resource/pubmed/commentcorrection/18191412-8895486, http://linkedlifedata.com/resource/pubmed/commentcorrection/18191412-9425080, http://linkedlifedata.com/resource/pubmed/commentcorrection/18191412-9790531
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
1096-0279
pubmed:author
pubmed:issnType
Electronic
pubmed:volume
58
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
257-62
pubmed:dateRevised
2010-10-4
pubmed:meshHeading
pubmed:year
2008
pubmed:articleTitle
Expression, purification and characterization of codon-optimized human N-methylpurine-DNA glycosylase from Escherichia coli.
pubmed:affiliation
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, LL level, S-122, 3800 Reservoir Road, NW, Washington, DC 20057, USA.
pubmed:publicationType
Journal Article, Research Support, N.I.H., Extramural