The cloning, expression, and purification of CDC14 using Escherichia coli

Abstract

Mitosis is a phase in the cell cycle when the replicated genetic information from a parent cell is separated and transmitted to its daughter cells. To ensure the genetic information is transmitted faithfully to the offspring, the cell monitors the progress in mitosis. In the late stage of mitosis, the activity of cyclin-dependent kinases (Cdks) is inhibited to complete and exit the mitosis. The phosphatase Cdc14p in budding yeast is a key component that counteracts the activity of Cdks and promotes mitotic exit. Cdc14p is regulated by the mitotic exit network (MEN) and the cell division cycle (CDC) fourteen early anaphase release (FEAR) network in budding yeast, and these two regulatory networks have been well studied at a genetic level. However, only a few studies explored the interactions of proteins in these two networks, and more investigations are necessary to understand these two regulatory networks at the protein level. We expressed the CDC14 gene in E. coli BL21 (DE3) strain, and purified the 6x histidine-tagged Cdc14p using a nickel metal affinity column. We also co-expressed the CDC14 with the CDC15 or CDC5 gene in the E. coli BL21 (DE3) strain, but no proteins were produced. An active form of Cdc14p was isolated by varying conditions for its production and purification. We are now working on the expression and purification of a kinase that will be used to study the interaction between the kinase and the Cdc14p in vitro.