Part:BBa_K2933151
His+Linker f+JOHN-1
This part encodes the fusion protein of His 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 162
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 51
Illegal PstI site found at 162 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 162
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 162
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 686
Usage and Biology
This composite part is made up with three basic parts, the HiS tag, the thrombin restriction site and our target protein JOHN-1. It encodes a protein which is JOHN-1 fused with His tag. The fusion protein is about 28.1 kD. It is convenient for us to purify our target protein.
Molecular cloning
First, we used the vector pET28a 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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