Part:BBa_K2933179
Tac promoter+RBS a+Linker g+GST+Linker e+IMP-71
This part consists of Tac promoter,RBS and protein coding sequence (GST+Linker e+IMP-71),and the biological module can be built into E.coli for protein expression.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1518
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1518
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1518
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1518
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 181
Usage and Biology
This composite part is made up with three basic parts(Tac promoter，RBS a and Linker g)and a composite part(GST+Linker e+IMP-71). It encodes a protein which is IMP-71 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 IMP-71 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 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 IMP-71. 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]Purification, crystallization and preliminary X-ray analysis of IMP-18, a class B carbapenemase from Pseudomonas aeruginosa.Furuyama T, Ishii Y, Ohya N, Tateda K, Hanson ND, Shimizu-Ibuka A.Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013 Dec;69(Pt 12):1397-400.
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