Construction And Application Of Sulfolobus Over-Expression Vector

It is important in expressing, purifying and analyzing crucial protein in vivo of solfataricus for the study on important life activities of extremely thermophilic archaea. The over-expression vector is constructed on the basis of pEXA vector inserted by araS promoter. Mrell with 6×His-tag in C-terminal are expressed successfully using it. Rad50 which interplay with Mrell is detected by co-purification. And it is ensured that the frame of complex is still unchanged in high concentration of salt (500mM NaCl). It indicates that the complex still keeps the function of DNA repair in adverse situation. Further analysis of co-purification make it clear that genome DNA fragments are the essential condition of foming the MR complex. In the absence of genome DNA fragments, the molecular chaperone of archaea may bind and protect Mrell. So this expression system possesses important sense in identifying the interact network of protein in vivo.The over-expression vector constructed in this research can not only be used to express important proteins, to identify important proteins and to study the in vivo role of the network, can also be used to analyze the function of archaeal Shine-Dalgarno which is the initiation component of translation. Archaeal translation mechanism is similar to bacteria, but the translation factors of archaea are similar to eukaryotes. Archaea has its own unique character. Therefore, the research of archaeal translation mechanism is helpful in understanding the evolution of species. So far direct study of Shine-Dalgarno in vivo is very few. In this research we constructed four plasmids named STlacS-pZC2, SDNllacS-pZC2, SDN21acS-pZC2 and SDN31acS-pZC2 on the ground of expression vector pZC2. The four plasmids can be directly used to analyze the function of Shine-Dalgarno sequence in vivo. Gene 3067 of S. solfataricus P2 owns inferred Shine-Dalgarno sequence. In SD sequence (GGTGTA), the first two bases, the middle two bases and the last two bases were respectively mutated in the three plasmids SDNllacS-pZC2, SDN21acS-pZC2 and SDN31acS-pZC2. And the lacS gene is used for reporter gene to analyze the impact of Shine-Dalgarno sequence on the translation efficiency. The results indicate that SD sequence is very important to the translation efficiency, and the critical GGTG is absolutely necessary. In this point, the archaeal translation mechanism is identical to bacteria. Research on the impression of Shine-Dalgarno sequence is very important to study the translation mechanism of leadless mRNA.