Part:BBa_K2933174
Tac promoter+RBS a+Linker g+GST+Linker e+TMB-2
This part consists of Tac promoter,RBS and protein coding sequence (GST+Linker e+TMB-2),and the biological module can be built into E.coli for protein expression.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 181
Usage and Biology
This composite part is made up with six basic parts(Tac promoter，RBS a , Linker g, GST, Linker e and TMB-2). It encodes a protein which is TMB-2 fused with GST tag. The fusion protein is about 53.2 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of TMB-2 and combine the two parts with the cutting site of Prescission Protease. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.
Molecular cloning
First, we used the vector pGEX-6p-1 and pET-28b_SUMO to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.
Figure 1. Left: The PCR result of TMB-2. Right: The verification results by enzyme digestion.
After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.
References
[1]Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding.Skagseth S, Christopeit T, Akhter S, Bayer A, Samuelsen Ø, Leiros HS.Antimicrob Agents Chemother. 2017 Jul 25;61(8).
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