The T7 RNA polymerase-regulated expression system shows strict selectivity and good transcriptional activity. Creative BioMart has combined the T7 RNA expression system with xylose operon Bacillus megaterium expression system to provide an efficient expression system for secretory and non-secretory expression. 50% or more of the total cellular protein can consist of the desired protein.
This technique combines T7 RNA polymerase and xylose operon for expression regulation. The expression system consists of two plasmids: pT7-RNAP and pPT7.
(1) a t7rnap gene controlled by a strong xylA promoter
(2) ampicillin and Chloramphenicol resistance genes, which are used for the selection in E. coli (Ampr) and Bacillus megaterium (CMR).
pPT7 is the shuttle vector in the system. It is responsible for t7rnap dependent expression of target genes. It contains
(1) a T7 promoter;
(2) a multi-clonal site downstream of the promoter with 10 unique restriction enzyme cleavage sites;
(3) ampicillin (E.coli) and tetracycline resistance (Bacillus megaterium) for selection
If the client is interested in the method of secretory protein expression, we will replace the pt7 vector with pPT7-SPLipA. This vector provides N-terminal signal peptide LipA, which secretes protein by Sec pathway.
Client who selects our expression service will need to submit your GOI sequence and detailed requirement online and our technical team will get in touch with you ASAP.
The team will work out the expression plan according to the requirements. After confirmation, we will start the expression procedure and report the progress of the project regularly. The given GOI sequence will be synthesized after codon analysis and optimization. Our services are quite flexible. Clients can choose to provide their own plasmids or/and strains or authorize us to buy from other companies. In general, our expression process is as follows:
| Services | Description |
| Cloning the DNA fragment of interest | The GOI is inserted into pPT7 vector and propagated in E.coli. |
| Transformation of B. megaterium protoplasts | For convenience, our protoplasts are pre-transformed with pT7-RNAP |
| Protein production | Test protein production Analysis of intracellular proteins Ammonium sulfate precipitation of proteins in the cell-free supernatant |
| Scale-up protein production | Grow larger culture and induce as indicated above Harvest cells at the time point of maximal protein production |
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