Cloning And Expression Of A Rab Gene From Euplotes Octocarinatus

Cellular organelles of eukaryotes have a distinctive spatial distribution and communicate through an elaborate system of vesicular-tubular transport. Rab proteins, behaving as molecular switches of vesicular trafficking, play an important role in different steps of vesicular trafficking. Ciliate, a unicellular protozoa and low eukaryotes in evolution, has perfect vesicular trafficking system among organelles. It's meaningful to study the function of Rab proteins in ciliates and further to explicit the mechanism of vesicular trafficking.The rob gene was amplified from macronuclear DNA of Euplotes octocarinatus. The size of gene was 783 bp long with an ORF of 624 bp encoding EoRabl protein and containing three in-frame TGA codes. The protein, putative belonging to Rab family, was characteristic of Rab proteins. Phylogenetic tree indicates the Rab protein may play a role in vesicular trafficking from endoplasmic reticulum to Golgi body.Two TGA in Eo-rabl were successfully mutated to TGC by site direct PCR procedure and the truncate Eo-rabl was obtained. Based on it, the third TGA was mutated to obtain the full-length Eo-rabl gene.Recombinant expression plasmids pGEX-Eorabl and pGEX-deEorabl were constructed. Recombinant genes of GST-EoRabl and GST-deEoRabl were highly expressed, respectively, in E. coli B121 (DE3) induced by IPTG. The results of Western blotting with anti-GST antibodies confirmed that putative fusion protein with molecular mass of 49 KD and 37 KD were the expected GST-EoRablfusion protein and truncated GST-deEoRab 1 fusion protein. GST-EoRabl fusion protein and truncated GST-deEoRabl fusion protein were purified by GST affinity chromatography and analyzed by cleaving with thrombin protease. The purity of EoRabl protein, cleaved GST-EoRabl protein with thrombin protease, reached 95 percent.