Propionate-inducible Vector Construction And Pro- Karyotic Expression Of Cyclic Di-nucleotide Synth- Etase Gene

Cyclic di-nucleotides are discovered as second messengers which played vital functions. Some studies have suggested that cyclic di-nucleotides could sense different extracellular messages to regulate many intracellular physiological and biochemical processes in various types of cells including bacterial biofilm formation and host innate immune response. As such, cyclic di-nucleotides have been developed into immunotherapy, immune prevention and vaccine adjuvants for the clinical treatment of human and animal dieeases. In connection with the importance of cyclic di-nucleotides in innate immune signaling as well as its potential applications, we constructed the prokaryotic expression plasmids of 10 different cyclic di-nucleotide synthetase from different species and expressed them by inducer in vitro. The present study aims to provide the basis for the further study of the synthesis of c-GAMP and c-di-GMP in vitro and for the furture research of vaccine adjuvant. Methods and results are as follows:Firstly, Bam Hâ… and Xhoâ… restriction enzymes were chosen for propionate-inducible vector construction of 10 different cyclic di-nucleotide synthetase by the comparison of nucleotide sequences between the multiple cloning site of the propionate-inducible vector and 10 different cyclic di-nucleotide synthetase genes. PCR and enzymatic digestion were used to get linear fragments of 10 cyclic di-nucleotide synthetase genes, which were then inserted into propionate-inducible vector followed by transformed into the E. coli BL21(DE3) strain. The recombinant plasmids were determined by restriction enzyme digestion and DNA sequencing. The expression of recombinant proteins induced by propionate was identified by SDS-PAGE analysis. Western blot analysis was used to test the soluble expression of recombinant proteins after ultrasonication. The results demonstrate that we have successfully constructed propionate-inducible prokaryotic expression plasmids containing 10 cyclic di-nucleotide synthetase genes. 8 of 10 recombinant plasmids could express in the soluble form in E. coli BL21.Secondly, VC0179 recombinant protein, which belongs to cyclic di-nucleotide synthetase in Vibrio cholerae, was purified through Ni-NTA purification system and cleaved by TEV protease. Moreover, c-di-GMP was enzymatically synthesized in vitro with therecombinant protein and detected by high performance liquid chromatography. After affinity purification with Ni-NTA, the high quality fusion protein was obtained. The VC0179 target protein could be generated by TEV cleavage. The synthesis of c-di-GMP in vitro by a one-step process demonstrated the recombinant protein with catalytic activity was obtained. These results suggested that this study will facilitate the further functional studies for adjuvant and high volume production of c-GAMP and c-di-GMP.