Cloning, Expression And Biological Function Of Sr-rex Gene From Streptomyces Rimosus M4018

Rex (Redox regulator, Rex) initially characterized in Streptomyces coelicolor is the first transcriptional regulator that responds directly to the poise of the NADH/NAD+redox,and it appears to be widespread among Gram-positive bacteria. As a novel mechanism of sensing intracellular redox state,the Rex regulatory system is not understood well.Therefore, the Rex from Streptomyces rimosus M4018, the oxytetracycline producer, was explored in this thesis, including its gene cloning, expression and biological function.The Sr-rex gene cloned from S. rimosus M4018's genome was sequenced and designated as Sr-rex.It was found that Sr-Rex was a new member of Rex protein family by BLAST in NCBI, and it had high homology with other Streptomyces strains,such as Streptomyces coelicolor A3(2) (84% identiy), Streptomyces avermitilis MA-4680 (84% identity), Streptomyces griseus(80% identity), Streptomyces lividans TK24 (71% indentity). Then, the Sr-rex gene was submitted to GenBank,and its accession number was GQ849479. DNA sequencing results suggested that Sr-rex encoded a protein of 281 amino acids with molecular weight (MW) of 29.2 kDa and isoelectric point (pI) of 5.17. Meanwhile, S.rimosus M4018 ROP (Rex operator) was also amplified with TaRaKa Genome Walking Kit. Its nucleotide sequence was the same as that of S. coelicolor ROP based on Sr-ROP DNA sequencing data.To obtain the Sr-Rex protein in vitro, the recombinant expression strain E.coli BL21(DE3) /pET28a-rex(his-Tag) was genetically constructed. Optimization of expression conditions for Sr-Rex production was carried out. The optimum expression conditions were as follows:the inducer IPTG was added with final concentration of 0.5 mmol/L when the cell optical density (OD600) reached about 0.5, and the IPTG induction duration was about 5h when the cells were grown at temperature of 37℃and at agitation speed of 220rpm. After cells harvest and lysis using ultrasonication, the Sr-Rex protein of 2.35mg/mLwith 99% purity was obtained through Ni-NTA affinity chromatography and DEAE anion-exchange chromatography.Finally, Sr-Rex binging ROP was tested by electrophoretic mobility shift assay (EMSA) between different ROP oligos and the purified Sr-Rex. The results revealed that both arms of the palindromic recognition sequence of ROP are neccesary for Sr-Rex:DNA interaction binding, and the core ROP nucleotide sequences with the maximum binding activity was: CATCTTTGTGCACGCGTTCACAAAGACA. In addition, it showed that the Rex-ROP binding activity was strongly inhibited by NADH.With increasing of the NADH concentration, the inhibitory effect on the Rex-Rop binding activity was enhanced, however, NAD+hardly had inhibitory effects on this binding activity.