Generation of an inducible system to express polo-like kinase, Cdc5 as TAP fusion protein during meiosis in Saccharomyces cerevisiae.
Tandem affinity purification (TAP) is a highly efficient method for isolation of protein complexes from endogenous biological macromolecules. TAP system consists of dual affinity tags that facilitates the sequential purification of the desired proteins expressed at their low levels in vivo. Polo-like kinases (PLK) are serine/threonine protein kinases that are the crucial regulators of cell cycle. Cdc5, the solitary PLK in budding yeast Saccharomyces cerevisiae, has diverse array of targets in cell cycle. The present study was undertaken to construct an estrogen-inducible system for expression of Cdc5-TAP to isolate the substrates of Cdc5 during meiosis, particularly, pachytene stage of meiosis I. Two yeast strains were constructed CDC5-IN (ndt80∆ pGAL1-CDC5-TAP) and Cdc5-kinase inactive mutant (ndt80∆ pGAL1-cdc5-N209A-TAP). The estrogen-inducible expression of Cdc5-TAP and cdc5-N209A-TAP was validated by Western analysis. The systems would serve as a valuable tool for purification of substrates binding to Cdc5-TAP by TAP affinity chromatography.
Faculty of Applied Sciences and Biotechnology, Shoolini University, Solan, Himachal Pradesh, 173212, India.
Rajni
R
Vaid
Faculty of Applied Sciences and Biotechnology, Shoolini University, Solan, Himachal Pradesh, 173212, India.
Kamal
K
Dev
Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, 20892, USA.
Michael
M
Lichten
Faculty of Applied Sciences and Biotechnology, Shoolini University, Solan, Himachal Pradesh, 173212, India. asourirajan@gmail.com.
Anuradha
A
Sourirajan
Cdc5; Estrogen inducible; Fusion protein; Joint molecules (JM); Kinase dead; Meiosis; Meiotic recombination; Pachytene; Polo-like kinase; Saccharomyces cerevisiae; Substrates; Tandem affinity purification