Vaid Rajni, Dev Kamal, Lichten Michael, Sourirajan Anuradha
Faculty of Applied Sciences and Biotechnology, Shoolini University, Solan, Himachal Pradesh, 173212, India.
Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, 20892, USA.
3 Biotech. 2016 Dec;6(2):185. doi: 10.1007/s13205-016-0503-x. Epub 2016 Aug 30.
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.
串联亲和纯化（TAP）是一种从内源性生物大分子中分离蛋白质复合物的高效方法。TAP系统由双亲和标签组成，有助于对体内低水平表达的所需蛋白质进行顺序纯化。Polo样激酶（PLK）是丝氨酸/苏氨酸蛋白激酶，是细胞周期的关键调节因子。Cdc5是芽殖酵母酿酒酵母中唯一的PLK，在细胞周期中有多种靶点。本研究旨在构建一种雌激素诱导系统，用于表达Cdc5-TAP，以分离减数分裂过程中，特别是减数第一次分裂粗线期Cdc5的底物。构建了两种酵母菌株CDC5-IN（ndt80∆ pGAL1-CDC5-TAP）和Cdc5激酶失活突变体（ndt80∆ pGAL1-cdc5-N209A-TAP）。通过蛋白质免疫印迹分析验证了Cdc5-TAP和cdc5-N209A-TAP的雌激素诱导表达。该系统将成为通过TAP亲和色谱法纯化与Cdc5-TAP结合的底物的有价值工具。
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