Ishii Jun, Izawa Keiko, Matsumura Shizuka, Wakamura Kanako, Tanino Takanori, Tanaka Tsutomu, Ogino Chiaki, Fukuda Hideki, Kondo Akihiko
Organization of Advanced Science and Technology, Kobe University, Japan.
J Biochem. 2009 Jun;145(6):701-8. doi: 10.1093/jb/mvp028. Epub 2009 Feb 23.
To allow the comprehensive assessments of yeast expression systems, a simple and immediate method for simultaneously evaluating the expression level and plasmid maintenance in yeast was demonstrated. This method uses green fluorescent protein (GFP) and flow cytometry (FCM) and is characterized by a dual analysis of the average intensity of GFP fluorescence and the population of GFP-expressing cells. The FCM analysis of GFP fluorescence intensity rapidly quantifies the expression level without complex manipulations, such as the enzymatic reaction of a lacZ reporter assay. Moreover, the single-cell analysis revealed that the proportion of cells expressing GFP in the cell cluster reflects the plasmid retention rate; therefore, the FCM analysis of the GFP-expressing population allows the immediate estimation of the plasmid retention rate without the 2- or 3-day incubation required for colony counting. We show that the FCM analysis with GFP reporter is a suitable method to explore the hopeful expression vector and host strain or establish the several expression systems exhibiting the characteristic properties in yeast.
为了能够对酵母表达系统进行全面评估，展示了一种用于同时评估酵母中表达水平和质粒维持情况的简单且直接的方法。该方法使用绿色荧光蛋白（GFP）和流式细胞术（FCM），其特点是对GFP荧光的平均强度和表达GFP的细胞群体进行双重分析。通过FCM对GFP荧光强度进行分析，无需诸如lacZ报告基因检测中的酶促反应等复杂操作，即可快速定量表达水平。此外，单细胞分析表明，细胞簇中表达GFP的细胞比例反映了质粒保留率；因此，对表达GFP的群体进行FCM分析可立即估计质粒保留率，而无需进行菌落计数所需的2至3天孵育。我们表明，使用GFP报告基因的FCM分析是探索有前景的表达载体和宿主菌株或建立在酵母中具有特征性质的多种表达系统的合适方法。
