The T7 RNA polymerase (T7 RNAP) expression system originates from the bacteriophage DNA-dependent RNA polymerase. In 1985, the first described T7 RNAP expression system was developed for Escherichia coli (Tabor and Richardson, 1985). Advantages of this system are the stringent selectivity and the high transcriptional activity so that it is possible to lead to a saturation of the protein-synthesizing machinery in E. coli. Consequently, 50% or more of the total cellular protein can consist of the desired protein (Studier and Moffatt, 1986).

The T7 RNAP expression system for B. megaterium combines the features of this system in E. coli with the above mentioned regulation by the xylose operon. This system is based on two parallel-replicating plasmids: pT7-RNAP and pPT7 (Gamer et al. 2009). In addition to the t7 rnap gene under control of the strong xylA promoter pT7-RNAP contains the genes for ampicillin and chloramphenicol resistance for easy selection in E. coli (AmpR) and B. megaterium (CmR). pPT7 is responsible for the T7 RNAP-dependent expression of the target gene. Downstream of the T7 promoter it comprises a multiple cloning site with ten unique restriction enzyme cleaving sites. Additionally the plasmid comprises two resistances against ampicillin (in E. coli) and tetracycline (in B. megaterium).

Next to the pPT7 vector, we offer an expression vector that leads to secretion of expressed proteins into the surrounding medium. pPT7-SPLipA provides the n-terminal signal peptide LipA that uses the Sec-pathway for protein secretion.

Furthermore, a great obstacle in protein production and engineering, the cloning of genes coding for proteins that are toxic to the cloning host E. coli, can be overcome by means of the T7 expression system due to the absence of T7 RNAP in the cloning host.

For your convenience MoBiTec offers B. megaterium protoplasts pretransformed with pT7-RNAP, so that you just have to insert your gene of interest into pPT7 and transform the pretransformed protoplasts with this plasmid.

For control purposes the gfp-expressing vector pPT7-GFP is included in the kit.

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