Burdychova Radka, Rychtera Milan, Horvath Radek, Dendis Milos, Bartos Milan
Faculty of Chemistry, Brno University of Technology, Purkynova 118, 612 00 Brno, Czech Republic.
FEMS Microbiol Lett. 2004 Jan 15;230(1):9-12. doi: 10.1016/S0378-1097(03)00622-0.
This study presents cloning and expression of Actinobacillus pleuropneumoniae Apx I toxin in Escherichia coli expression system to produce fusion protein for the subsequent immunological studies. The gene coding Apx I toxin was amplified from the A. pleuropneumoniae serotype 10 DNA using polymerase chain reaction and cloned to vector under the control of strong, inducible T7 promoter. The presence of insert was confirmed by PCR screening and sequencing after the propagation of recombinant DNA in E. coli cells. The gene coding A. pleuropneumoniae Apx I toxin was extended with a segment to encode a polyhistidine tag linked to its C-terminal sequence allowing a one-step affinity purification of the complex with Ni-NTA resin. Expression of the Apx I coding sequence in E. coli resulted in the formation of insoluble inclusion bodies purified according to a standard purification protocol. The ease of this expression system, the powerful single-step purification and low costs make it possible to produce Apx I in large amounts to further study the role of Apx I in physiological processes.
本研究展示了胸膜肺炎放线杆菌Apx I毒素在大肠杆菌表达系统中的克隆与表达，以生产融合蛋白用于后续的免疫学研究。使用聚合酶链反应从胸膜肺炎放线杆菌血清型10的DNA中扩增编码Apx I毒素的基因，并将其克隆到在强诱导性T7启动子控制下的载体中。在重组DNA在大肠杆菌细胞中繁殖后，通过PCR筛选和测序确认插入片段的存在。编码胸膜肺炎放线杆菌Apx I毒素的基因在其C端序列上延伸了一段以编码与多组氨酸标签相连的片段，从而允许使用Ni-NTA树脂对复合物进行一步亲和纯化。Apx I编码序列在大肠杆菌中的表达导致形成了根据标准纯化方案纯化的不溶性包涵体。这种表达系统的简便性、强大的单步纯化能力和低成本使得大量生产Apx I成为可能，以便进一步研究Apx I在生理过程中的作用。
