Construction And Application Of Over-Expression Vectors And Tag Vectors In Streptomvcetes

Streptomycetes are Gram-positive, soil-dwelling microorganisms, which can produce many kinds of antibiotics, enzymes, immuno-suppressives, etc., with commercial and academic value. In recent years, it has been developed as the hosts for the expression of exogenous proteins. In this study, two high-copy number vectors, pL97and pL98, that contain the strong constitutive ermEp*and ssrA promoter (ssrAp), respectively, and an inducible high-copy vector, pL99, with the PnitA-NitR system were constructed based on pIJ8668backbone and the pIJ101replicon. These vectors have pUC18and pIJ101replication origins for high-copy plasmid number in Escherichia coli and Streptomycetes, respectively, and the oriT (RK2) allows the efficient and convenient plasmid transfer from E. coli to Streptomycetes. The transformants can be easily selected with apramycin. There is also a multiple cloning site (MCS) between promoter and terminator convenient for exogenous gene cloning. The enhanced green fluorescent protein (EGFP) gene and redD, a pathway-specific regulatory gene for the production of undecylprodigiosin in S. coelicolor, were inserted into these plasmids and could be over-expressed in S. coelicolor. Western blot revealed that the expression of EGFP was dramatically increased compared to the cell with a single copy of EGFP. Furthermore, the production of undecylprodigiosin was also greatly enhanced in the cells constitutively over-expressing redD. Hence, the high-copy plasmids could be readily used to express foreign proteins and for production of secondary metabolites, especially the antibiotics, potentially for industrial applications.After years of research, Streptomycetes genetics have made considerable development. However, not much information is available about the intracellular signal transduction based on protein-protein or DNA-protein interactions. In this study, a strong constitutive promoter ermEp*was inserted into promoter-probe plasmid pIJ8660followed by a multiple cloning site instead of the gene egfp. Then we introduced3FLAG,3HA,13Myc,3Strep-tagⅡ,18His tags through codon optimization into the downstream of promoter and constructed a series of vectors containing a single tag or two tags. Then we successfully expressed the ECF sigma factor SigT with the fused tags in Streptomyces coelicolor. Basing on this, we identified the proteins interacting with SigT, including the anti-sigma factor RstA using the FLAG-His tandem affinity purification (TAP) combined with the method of LC-MS. Construction of series of tagged vectors which made a convenience for proteins expression, identification and purification laid the foundation for the study of protein-protein interactions and protein-DNA interactions in vivo and provided a powerful tool for screening the related regulatory elements and targets at the level of genome and proteome by TAP and ChIP-Seq methods.