Part:BBa_K2933128
His+Linker a+Sumo+Linker b+CPS-1
This part encodes the fusion protein of His tag, sumo tag and CPS-1 to promote the expression and purification of target protein(CPS-1).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 256
Illegal PstI site found at 892 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 256
Illegal NheI site found at 33
Illegal PstI site found at 892 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 256
Illegal BglII site found at 145
Illegal BglII site found at 1174
Illegal BamHI site found at 344 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 256
Illegal PstI site found at 892 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 256
Illegal PstI site found at 892
Illegal AgeI site found at 793 - 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 CPS-1. It encodes a protein which is CPS-1 fused with His-Sumo tag. The fusion protein is about 45.2kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of CPS-1 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]Dereje Dadi Gudeta, Valeria Bortolaia,The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing β-Lactamases of Potential Clinical Relevance[J]，Antimicrobial Agents and chemotherapy,January 2016
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.
Figure 1. a: The PCR result of CPS-1. b: 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.
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