Part:BBa_K2933140
His+Linker a+Sumo+Linker b+Fla.103
This part encodes the fusion protein of His tag, sumo tag and Fla.103 to promote the expression and purification of target protein(Fla.103).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 256
Illegal PstI site found at 431 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 256
Illegal NheI site found at 33
Illegal PstI site found at 431 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 256
Illegal BglII site found at 145
Illegal BglII site found at 913
Illegal BamHI site found at 344 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 256
Illegal PstI site found at 431 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 256
Illegal PstI site found at 431 - 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 Fla.103. It encodes a protein which is Fla.103 fused with His-Sumo tag. The fusion protein is about 40.7kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of Fla.103 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
Molecular cloning
First, 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.We then extracted plasmids and performed double enzyme digestion verification.
Figure 1. (a) The PCR result of Fla.103. (b) The verification results by enzyme digestion.The 1 is the original plasmid.The 2 is the
results verified by double 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.
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