Part:BBa_K2933113
GST+Linker+JOHN-1
This part encodes the fusion protein of GST tag and JOHN-1 to promote the expression and purification of target protein(JOHN-1).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 774
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 774
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 774
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 774
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 85
Illegal SapI.rc site found at 1298
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 JOHN-1. It encodes a protein which is JOHN-1 fused with GST tag. The fusion protein is about 54.1 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of JOHN-1 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 JOHN-1. 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]Naas Thierry,Bellais Samuel,Nordmann Patrice. Molecular and biochemical characterization of a carbapenem-hydrolysing beta-lactamase from Flavobacterium johnsoniae.[J]. Journal of Antimicrobial Chemotherapy,2003,51(2).
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