Linked Life Data

20699085

Source:http://linkedlifedata.com/resource/pubmed/id/20699085

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2010-9-29
pubmed:abstractText
Aurora B kinase plays a critical role in regulating mitotic progression, and its dysregulation has been linked to tumorigenesis. The structure of the kinase domain of human Aurora B and the complementary information of binding thermodynamics of known Aurora inhibitors is lacking. Towards that effort, we sought to identify a human Aurora B construct that would be amenable for large-scale protein production for biophysical and structural studies. Although the designed AurB(69-333) construct expressed at high levels in Escherichia coli, the purified protein was largely unstable and prone to aggregation. We employed thermal-shift assay for high-throughput screening of 192 conditions to identify optimal pH and salt conditions that increased the stability and minimized aggregation of AurB(69-333). Direct ligand binding analyses using temperature-dependent circular dichroism (TdCD) and TR-FRET-based Lanthascreen™ binding assay showed that the purified protein was folded and functional. The affinity rank-order obtained using TdCD and Lanthascreen™ binding assay correlated with enzymatic IC50 values measured using full-length Aurora B protein for all the inhibitors tested except for AZD1152. The direct binding results support the hypothesis that the purified human AurB(69-333) fragment is a good surrogate for its full-length counterpart for biophysical and structural analyses.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
1096-0384
pubmed:author
pubmed:copyrightInfo
Copyright © 2010 Elsevier Inc. All rights reserved.
pubmed:issnType
Electronic
pubmed:day
15
pubmed:volume
503
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
191-201
pubmed:dateRevised
2011-7-11
pubmed:meshHeading
pubmed-meshheading:20699085-Amino Acid Sequence, pubmed-meshheading:20699085-Circular Dichroism, pubmed-meshheading:20699085-Cloning, Molecular, pubmed-meshheading:20699085-Enzyme Stability, pubmed-meshheading:20699085-Escherichia coli, pubmed-meshheading:20699085-Fluorescence Resonance Energy Transfer, pubmed-meshheading:20699085-Humans, pubmed-meshheading:20699085-Inhibitory Concentration 50, pubmed-meshheading:20699085-Mass Spectrometry, pubmed-meshheading:20699085-Molecular Sequence Data, pubmed-meshheading:20699085-Molecular Weight, pubmed-meshheading:20699085-Protein Folding, pubmed-meshheading:20699085-Protein Structure, Tertiary, pubmed-meshheading:20699085-Protein-Serine-Threonine Kinases, pubmed-meshheading:20699085-Recombinant Fusion Proteins, pubmed-meshheading:20699085-Sequence Homology, Amino Acid, pubmed-meshheading:20699085-Thermodynamics
pubmed:year
2010
pubmed:articleTitle
Expression, purification, stability optimization and characterization of human Aurora B kinase domain from E. coli.
pubmed:affiliation
Protein Science Department, Cambridge, MA 02141, USA. payal.sheth@spcorp.com
pubmed:publicationType
Journal Article, Comparative Study