Méndez Andrea A E, Pena Liliana B, Curto Lucrecia M, Sciorra Marcelo D, Ulloa Rita M, Garza Aguilar Sara M, Vázquez Ramos Jorge M, Gallego Susana M
Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto de Química y Fisicoquímica Biológicas "Profesor Alejandro C, Paladini" (IQUIFIB), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
Instituto de Investigaciones en Ingeniería Genética y Biología Molecular D, Héctor N, Torres (INGEBI), Argentina.
Protein Expr Purif. 2020 Jan;165:105483. doi: 10.1016/j.pep.2019.105483. Epub 2019 Aug 31.
The complex formed by the cyclin-dependent kinase A (CDKA) and cyclin D is responsible for the G1-S transition in the plant cell cycle. Maize (Zea mays L) CDKA; 1 and CycD6; 1 were cloned and expressed in E. coli. The present study describes the optimization of both proteins production using a statistical approach known as response surface methodology (RSM). The experimental design took into account the effects of four variables: optical density of the culture (OD) before induction, isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration, post-induction temperature, and post-induction time. For each protein, a 2 full factorial central composite rotary design for these four independent variables (at five levels each) was employed to fit a polynomial model; which indicated that 30 experiments were required for this procedure. An optimization of CDKA; 1 and CycD6; 1 production levels in the soluble fraction was achieved. Protein conformation and stability were studied by circular dichroism and fluorescence spectroscopy. Finally, in vitro Cyc-CDK complex formation and its kinase activity were confirmed.
细胞周期蛋白依赖性激酶A（CDKA）和细胞周期蛋白D形成的复合物负责植物细胞周期中的G1-S转换。克隆了玉米（Zea mays L）的CDKA;1和CycD6;1，并在大肠杆菌中表达。本研究描述了使用称为响应面法（RSM）的统计方法对这两种蛋白质的生产进行优化。实验设计考虑了四个变量的影响：诱导前培养物的光密度（OD）、异丙基β-D-1-硫代半乳糖苷（IPTG）浓度、诱导后温度和诱导后时间。对于每种蛋白质，采用这四个独立变量（每个变量有五个水平）的2全因子中心复合旋转设计来拟合多项式模型；这表明该过程需要进行30次实验。实现了CDKA;1和CycD6;1在可溶级分中生产水平的优化。通过圆二色性和荧光光谱研究了蛋白质的构象和稳定性。最后，证实了体外Cyc-CDK复合物的形成及其激酶活性。
