Characteristics Of Solfataricus Over-expression Vector And Transcription Regulation Of AraS Operon In Sulfolobus Solfataricus

The expression of thermophilic proteins is not a fresh barrier in front of researchers all over the world who put their energy into the high level research about some proteins which may play an important role in some life progresses, such as DNA repair, Transcription, Translation, and CRISPR. There are some major problems:lack of mature genetic protein express system; low level of objective products when proteins are expressed in homologous hosts; inclusion body are easily formed in heterologous hosts, like E. coli. To slove these problems, researchers in our lab has constructed a series of over-expression Vectors (pEXA, paraS1, paraS2, p10b, p7d and pSeSD) based on a E. coli-Sulfolobus shuttle vector pZC1. These over-expression can confers high levels of recombinant protein expression in hyperthermophlic Archaeon Sulfolobus islandicus. In this research we identified a ParaS-SD combination directed a robust expression of objective protein. ParaS-SD contains a araS promoter and a RBS in the proper location upstream of translation start codon. And we also found that ATG and GTG are the most favorite and most abhorrent translation start codon of hyperthermophlic Archaeon Sulfolobus islandicus.araS operon is a four gene(araS/araT/araU/araV) contained operon which can induced by arabinose. It’s products are araS(arabinose combined protein), araT(membrane permeability protein), araU (membrane permeability protein), araV(possess ATPase activity). There are only limited numbers of operon in the genome of Sulfolobus islandicus and research results about the transcription and translation mechanisms of operon in Archaeon. As we know, Archaeon is familar with eukaryote in processions of DNA process, so it’s very meaningful to carry out research works in operon’s expression.In the base of our β-galactosidase activity data and RT-PCR data, we reported that there is no inner promoter in araS operon of Sulfolobus solfataricus, and araS/araT/araU/araV are co-transcripted on a single mRNA. But further real-time PCR data shows that transcripts number of araS are almost9times more than araT’s in one Sulfolobus solfataricus cell. This indicate that there must be a novel mechanism which can reduce transcription efficiency greatly. Obviously, the untranslated nucleic acid residues between araS and araT may be the most possible reason which resulted in that wired phenomenon, because this UTR contains35bp and A/T rich. But when we take the UTR to BLASTn, there is no similar residues exist. Then we aligned all the UTR of operons in Sulfolobus solfataricus, no distinct features was got after the alignment. There is only one feature:they are all A/T rich. So we suggest that this element do not have any sepuence conservatism, but have the similar secondary structure conservatism with terminator.