Renesto P, Raoult D
Unité des Rickettsies, CNRS-UMRA-6020, Faculté de Médecine, 13480 Marseille, France.
Ann N Y Acad Sci. 2003 Jun;990:642-52. doi: 10.1111/j.1749-6632.2003.tb07439.x.
The availability of the complete genome sequences of several organisms allows the comparative analysis of genomes, a branch of bioinformatics known as genomics. With this approach, much can be learned about the biology of organisms that are difficult to culture, even when few, if any, of their proteins have been isolated and studied directly. We have focused our interest on Rickettsia conorii, an obligate intracellular bacterium responsible for Mediterranean spotted fever, a disease endemic in southern Europe. While bioinformatic annotation of the complete genome of this bacteria has allowed identification of 1,374 genes, a large number of them remain functionally uncharacterized. The final goal of many experiments in molecular biology is to use biological systems to synthesize the protein encoded by the gene being studied. Because three-dimensional structures are more resilient to evolution and change than amino acid sequences, structure determination of some open reading frames should also exhibit structural similarity to previously described protein families. We have thus initiated a systematic expression and structure determination program for the proteins encoded by rickettsial genes of interest. We have cloned different genes of R. conorii by recombinational cloning (GATEWAY), Invitrogen) a method that uses in vitro site-specific recombination to accomplish a directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency. The constructions in p-Dest17 yielded several clones able to express recombinant proteins with a C-terminal histidine tag. Expression of corresponding proteins was then performed using a cell-free protein expression system (Rapid Translation System, RTS, Roche Diagnostics). The recombinational cloning approach coupled to RTS provides an approach to rapid optimization of protein expression and is very useful to express rickettsial proteins. Moreover, this system is able to overcome some of the limitations encountered with rickettsial proteins highly toxic for E. coli or insect cells.
几种生物完整基因组序列的可得性使得基因组的比较分析成为可能，这是生物信息学的一个分支，称为基因组学。通过这种方法，可以了解许多难以培养的生物的生物学特性，即使它们几乎没有（如果有的话）蛋白质被直接分离和研究过。我们将兴趣集中在康氏立克次体上，它是一种专性细胞内细菌，可引起地中海斑疹热，这种疾病在欧洲南部流行。虽然对这种细菌的完整基因组进行生物信息学注释已确定了1374个基因，但其中许多基因的功能仍未得到表征。分子生物学中许多实验的最终目标是利用生物系统合成所研究基因编码的蛋白质。由于三维结构比氨基酸序列更能抵抗进化和变化，一些开放阅读框的结构测定也应显示出与先前描述的蛋白质家族的结构相似性。因此，我们启动了一个针对感兴趣的立克次体基因编码的蛋白质的系统表达和结构测定计划。我们通过重组克隆（GATEWAY，Invitrogen）克隆了康氏立克次体的不同基因，该方法利用体外位点特异性重组来高效完成PCR产物的定向克隆以及随后将DNA片段自动亚克隆到新的载体骨架中。在p-Dest17中的构建产生了几个能够表达带有C末端组氨酸标签的重组蛋白的克隆。然后使用无细胞蛋白质表达系统（快速翻译系统，RTS，罗氏诊断公司）进行相应蛋白质的表达。重组克隆方法与RTS相结合提供了一种快速优化蛋白质表达的方法，对于表达立克次体蛋白非常有用。此外，该系统能够克服立克次体蛋白对大肠杆菌或昆虫细胞具有高毒性所带来的一些限制。
