Badri M Amine, Rivard Daniel, Coenen Karine, Vaillancourt Louis-Philippe, Goulet Charles, Michaud Dominique
Département de phytologie, CRH/INAF, Université Laval, Québec, Canada.
Proteomics. 2009 Jan;9(2):233-41. doi: 10.1002/pmic.200700233.
We describe a SELDI-TOF MS procedure for the rapid detection and quantitation of low-molecular-weight recombinant proteins expressed in plants. Transgenic lines of potato (Solanum tuberosum L.) expressing the clinically useful protein bovine aprotinin or the cysteine protease inhibitor corn cystatin II were generated by Agrobacterium tumefaciens-mediated transformation, and then used as test material for the analyses. Real-time RT-PCR amplifications and detection of the recombinant proteins by immunoblotting were first conducted for transformed potato lines accumulating the proteins in different cell compartments. Both proteins were found at varying levels in leaves, depending on their final cellular destination and transgene expression rate. These conclusions drawn from standard immunodetection assays were easily confirmed by SELDI-TOF MS comparative profiling, after immobilizing the leaf proteins of control and transformed lines on protein biochips for weak cationic exchange. This procedure, carried out in less than 2 h, allows for the rapid comparison of recombinant protein levels in transgenic plant lines. The molecular weight of immobilized proteins can also be determined directly from the MS spectra, thus providing a simple way to assess the structural integrity and homogeneity of recombinant proteins in planta, and to identify the most suitable cellular compartments for their heterologous production.
我们描述了一种用于快速检测和定量植物中表达的低分子量重组蛋白的表面增强激光解吸电离飞行时间质谱（SELDI-TOF MS）方法。通过根癌农杆菌介导的转化产生了表达临床上有用的蛋白质牛胰蛋白酶抑制剂或半胱氨酸蛋白酶抑制剂玉米胱抑素II的转基因马铃薯品系（Solanum tuberosum L.），然后将其用作分析的测试材料。首先对在不同细胞区室中积累蛋白质的转化马铃薯品系进行实时逆转录聚合酶链反应（RT-PCR）扩增和通过免疫印迹法检测重组蛋白。根据它们最终的细胞定位和转基因表达率，在叶片中发现这两种蛋白质的含量各不相同。在将对照品系和转化品系的叶片蛋白固定在用于弱阳离子交换的蛋白质生物芯片上后，通过SELDI-TOF MS比较分析很容易证实从标准免疫检测分析得出的这些结论。该过程在不到2小时内完成，可快速比较转基因植物品系中重组蛋白的水平。固定化蛋白的分子量也可以直接从质谱图中确定，从而提供一种简单的方法来评估植物中重组蛋白的结构完整性和均一性，并确定其异源生产最合适的细胞区室。
