Quantitative Y2H screening: Cloning and signal peptide engineering of a fungal secretory LacA gene and its application to yeast two-hybrid system as a quantitative reporter
Quantitative Y2H screening: Cloning and signal peptide engineering of a fungal secretory LacA gene and its application to yeast two-hybrid system as a quantitative reporter
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DOI：
10.1016/j.jbiotec.2010.02.007
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发表时间：
2010-04-15
影响因子：
4.1
通讯作者：
Kawarasaki, Yasuaki
中科院分区：
文献类型：
--
作者：
Kamiya, Takuma;Ojima, Teruyo;Kawarasaki, Yasuaki
将真菌分泌型β-半乳糖苷酶基因LacA 3引入酵母双杂交系统，建立了一个适合高通量筛选的蛋白质/肽定量筛选系统。我们描述了原始真菌LacA基因的分子克隆和信号肽优化，该基因的胞外表达最初对宿主细胞有毒。工程化的LacA，LacA 3，表现出较低的毒性，从而改善了宿主的培养特性。在一定的诱导条件下，该酶释放到培养基中的量与细胞密度有关，与生长期无关。释放的酶保持了野生型性质，是高度糖基化的，在宽pH范围和高温下稳定，并具有酸性pH最适。在具有新型报道基因与常规Y2 H报道基因组合的Y2 H系统中，根据相互作用强度，酵母菌落在生长环境中可见地染成蓝色、白色或红色。由于细胞外LacA 3表达增加，具有更稳定相互作用的克隆很容易被发现为具有更大蓝色晕圈的菌落。利用该报告基因对cDNA文库进行了高通量的Y2 H筛选。还举例说明了新的Y2 H系统在肽段定向进化中的应用。(C)2010 Elsevier BV保留所有权利。
A quantitative protein/peptide screening system amenable to high-throughput screening has been developed by furnishing conventional yeast two-hybrid (Y2H) system with an engineered fungal secretory beta-galactosidase gene (designated LacA3). We describe the molecular cloning and signal peptide-optimization of the original fungal LacA gene of which extracellular expression was initially toxic to the host cell. The engineered LacA, LacA3, showed less toxicity, resulting in improved cultural properties of the host. The release of the enzyme to the medium was constant to the cell density under a certain induction condition and independent of the growth phase. The released enzyme kept the wild type properties, was highly glycosylated, stable in a wide pH range and high temperature, and had an acidic pH optimum. In the Y2H system with the novel reporter in combination with the conventional Y2H reporters, the yeast colonies are visibly stained in blue, white or red in the growth context, according to the interaction intensity. The clones with the more stable interactions are easily found as colonies with the larger blue halos, due to the increased extracellular LacA3 expression. A quantitative, high-throughput Y2H screening of cDNA library based on the novel reporter was demonstrated. An application of the novel Y2H system to directed evolution of a peptide fragment was also exemplified. (C) 2010 Elsevier B.V. All rights reserved.