Falk Ronny, Agaton Charlotta, Kiesler Eva, Jin Shaobo, Wieslander Lars, Visa Neus, Hober Sophia, Ståhl Stefan
Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), SE-106 91 Stockholm, Sweden.
Biotechnol Appl Biochem. 2003 Dec;38(Pt 3):231-9. doi: 10.1042/BA20030091.
A novel, improved dual bacterial-expression system, designed for large-scale generation of high-quality polyclonal antibody preparations intended for proteomics research, is presented. The concept involves parallel expression of cDNA-encoded proteins, as a fusion with two different tags in two separate vector systems. Both systems enable convenient blotting procedures for expression screening on crude bacterial cell cultures and single-step affinity purification under denaturing conditions. One of the fusion proteins is used to elicit antibodies, and the second fusion protein is used in an immobilized form as an affinity ligand to enrich antibodies with selective reactivity to the cDNA-encoded part, common for the two fusion proteins. To evaluate the system, four cDNA clones from putative nuclear proteins from the non-biting midge Chironomus tentans were expressed. Antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting and immunofluorescence procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The four antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the four antibodies gave specific staining in Western-blot analysis of nuclear cell extracts. Furthermore, two of the antibody preparations gave specific staining in immunofluorescence analysis of C. tentans cells. We conclude that the dual-vector concept presented offers a highly stringent strategy for the generation of monospecific polyclonal antibodies, which are useful in proteomics research.
本文介绍了一种新型的、经过改进的双细菌表达系统，该系统旨在大规模生产用于蛋白质组学研究的高质量多克隆抗体制剂。其概念是在两个独立的载体系统中，将cDNA编码的蛋白质与两种不同的标签融合进行平行表达。这两个系统都能在粗细菌细胞培养物上方便地进行印迹程序以进行表达筛选，并在变性条件下进行单步亲和纯化。其中一种融合蛋白用于产生抗体，第二种融合蛋白以固定化形式作为亲和配体，用于富集对两种融合蛋白共有的cDNA编码部分具有选择性反应性的抗体。为了评估该系统，表达了来自非吸血摇蚊Chironomus tentans假定核蛋白的四个cDNA克隆。针对这些cDNA编码蛋白产生、富集了抗体，并将其用于印迹和免疫荧光程序，以确定与cDNA相对应的天然蛋白的表达模式。这四种抗体制剂对相应的重组cDNA编码蛋白表现出特异性反应，并且在核细胞提取物的蛋白质印迹分析中，四种抗体中的三种给出了特异性染色。此外，在Chironomus tentans细胞的免疫荧光分析中，两种抗体制剂给出了特异性染色。我们得出结论，所提出的双载体概念为产生单特异性多克隆抗体提供了一种高度严格的策略，这些抗体在蛋白质组学研究中很有用。
