Part:BBa_K2933121
GST+Linker e+IMP-71
This part encodes the fusion protein of GST tag and IMP-71 to promote the expression and purification of target protein(IMP-71).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1422
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1422
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1422
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1422
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 85
Usage and Biology
This composite part is made up with three basic parts, the GST tag, the cutting site of Prescission Protease and our target protein 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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