Fasolo Joseph, Im Hogune, Snyder Michael P
Department of Genetics, Stanford University.
Department of Genetics, Stanford University; Stanford Center for Genomics and Personalized Medicine, Stanford University;
J Vis Exp. 2015 Aug 2(102):e51872. doi: 10.3791/51872.
High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis to determine a high confidence set of protein interactions for downstream validation in vivo.
使用含有V5表位标签用于读出的亲和纯化酵母激酶融合蛋白，对包含约4200种重组酵母蛋白的高密度功能蛋白微阵列进行激酶蛋白-蛋白相互作用检测。纯化的激酶是通过培养一种为高拷贝蛋白生产优化的酵母菌株获得的，该菌株携带一个在GAL诱导型启动子下含有激酶-V5融合构建体的质粒。酵母在含有中性碳源的限制性培养基中生长6小时，然后用2%半乳糖诱导。接下来，收获培养物并使用标准亲和色谱技术纯化激酶，以获得用于该检测的高度纯化的蛋白激酶。将纯化的激酶用激酶缓冲液稀释至适合该检测的范围，并在与蛋白微阵列杂交之前封闭蛋白微阵列。杂交后，用单克隆V5抗体探测阵列，以鉴定被激酶-V5蛋白结合的蛋白。最后，使用标准微阵列扫描仪扫描阵列，并提取数据用于下游信息学分析，以确定一组高可信度的蛋白相互作用，用于体内下游验证。
