Tri-cistronic cloning, overexpression and purification of human Rad9, Rad1, Hus1 protein complex.
The least understood components of the DNA damage checkpoint are the DNA damage sensors. Genetic studies of Schizosaccharomyces pombe identified six yeast genes, Rad3, Rad17, Rad9, Rad1, Hus1, and Rad26, which encode proteins thought to sense DNA damage and activate the checkpoint-signaling cascade. It has been suggested that Rad9, Rad1 and Hus1 make a heterotrimeric complex forming a PCNA-like structure. In order to carry out structural and biophysical studies of the complex and its associated proteins, the cDNAs encoding full length human Rad9, Rad1 and Hus1 were cloned together into the pET28a vector using a one-step ligation procedure. Here we report successful tri-cistronic cloning, overexpression and purification of this three-protein complex using a single hexa-histidine tag. The trimeric protein complex of Rad9, Rad1 and Hus1 was purified to near homogeneity, yielding approximately 10mg of protein from one liter of Escherichia coli culture.
Singh
Vinay Kumar
VK
Department of Biochemistry, Queen's University, Kingston, Ont., Canada K7L 3N6.
Nurmohamed
Salima
S
Davey
Scott K
SK
Jia
Zongchao
Z
D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005092:Exonucleases / Q000235:genetics* / Q000302:isolation & purification; D005822:Genetic Vectors; D046911:Macromolecular Substances / Q000302:isolation & purification
D018797:Cell Cycle Proteins; C549605:HUS1 protein, human; C462949:HUS1B protein, human; D046911:Macromolecular Substances; C069930:rad9 protein; D005092:Exonucleases; C113574:Rad1 protein, human