Part:BBa_K2933133
His+Linker a+Sumo+Linker b+BcII-194
This part encodes the fusion protein of His tag, sumo tag and BcII-194 to promote the expression and purification of target protein(BcII-194).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 256
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 256
Illegal NheI site found at 33 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 256
Illegal BglII site found at 145
Illegal BamHI site found at 344 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 256
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 256
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This composite part is made up with five basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein BcII. It encodes a protein which is BcII fused with His-Sumo tag. The fusion protein is about 40.1kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of BcII and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.
References
[1]Sung-Kun Kim, Mara Demuth, Sara R. Schlesinger, Sung Joon Kim, Jonathan Urbanczyk, Robert W. Shaw & Hyunshun Shin (2016) Inhibition of Bacillusanthracis metallo-β- lactamase by compounds with hydroxamic acid functionality, Journal of Enzyme Inhibition and Medicinal Chemistry, 31:sup4, 132-137, DOI: 10.1080/14756366.2016.1222580
Molecular cloning
First,we obtained BcII by PCR.
Figure 1. The PCR result of BcII.
Then we used the vector pET28b-sumo to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.
Figure 2. Left:The plasmid of BcII.Right:The verification results by enzyme digestion.
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