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Identification and purification of a soluble region of BubR1: a critical component of the mitotic checkpoint complex

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Authors
Yoon, Jongchul; Kang, Yup; Kim, Kyunggon; Park, Jungeun; Kim, Youngsoo
Issue Date
2005-06-11
Publisher
Elsevier
Citation
Protein Expr Purif. 2005 Nov;44(1):1-9.
Keywords
Amino Acid Motifs/geneticsAmino Acid Sequence*Anaphase/physiologyAnimalsCell Cycle Proteins/metabolismChromosome Segregation/physiologyCloning, Molecular/methodsGenetic Vectors/geneticsMolecular Sequence DataProtein Binding/physiologyProtein Kinases/*genetics/*isolation & purification/metabolismProtein Structure, TertiaryProtein-Serine-Threonine KinasesRatsSolubility
Abstract
The mitotic checkpoint complex (MCC) ensures the fidelity of chromosomal segregation, by delaying the onset of anaphase until all sister chromatids have been properly attached to the mitotic spindle. In essence, this MCC-induced delay is achieved via the inhibition of the anaphase-promoting complex (APC). Among the components of the MCC, BubR1 plays two major roles in the functions of the mitotic checkpoint. First, BubR1 is able to inhibit APC activity, either by itself or as a component of the MCC, by sequestering a APC coactivator, known as Cdc20. Second, BubR1 activates mitotic checkpoint signaling cascades by binding to the centromere-associated protein E, a microtubule motor protein. Obtaining highly soluble BubR1 is a prerequisite for the study of its structure. BubR1 is a multi-domain protein, which includes a KEN box motif, a mad3-like region, a Bub3 binding domain, and a kinase domain. We obtained a soluble BubR1 construct using a three-step expression strategy. First, we obtained two constructs from BLAST sequence homology searches, both of which were expressed abundantly in the inclusion bodies. We then adjusted the lengths of the two constructs by secondary structure prediction, thereby generating partially soluble constructs. Third, we optimized the solubility of the two constructs by either chopping or adding a few residues at the C-terminus. Finally, we obtained a highly soluble BubR1 construct via the Escherichia coli expression system, which allowed for a yield of 10.8 mg/L culture. This report may provide insight into the design of highly soluble constructs of insoluble multi-domain proteins.
ISSN
1046-5928 (Print)
Language
English
URI
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15946858

http://dx.dor.org/10.1016/j.pep.2005.04.020

http://hdl.handle.net/10371/47070
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College of Medicine/School of Medicine (의과대학/대학원)Molecular and Genomic Medicine (분자유전체의학전공)Journal Papers (저널논문_분자유전체의학전공)
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