Identification and purification of a soluble region of BubR1: a critical component of the mitotic checkpoint complex.
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.
Yoon
Jongchul
J
Division of Molecular Genomic Medicine, College of Medicine, Seoul National University, Yongon-Dong, Seoul 110-799, Korea.
Kang
Yup
Y
Kim
Kyunggon
K
Park
Jungeun
J
Kim
Youngsoo
Y
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000705:Anaphase / Q000502:physiology; D000818:Animals; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D020090:Chromosome Segregation / Q000502:physiology; D003001:Cloning, Molecular / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000502:physiology; D011494:Protein Kinases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D051381:Rats; D012995:Solubility
D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; C577488:CDC20 protein, human; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases