Part:BBa_K2933251
RBS b+Linker h+His+Linker a+Sumo+Linker b+CPS-1+T7 terminator
This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+CPS-1) and T7 terminator,and the biological module can be build into E.coli for protein expression. This part can be prefaced with promoters of different strengths and types to regulate expression function.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 298
Illegal PstI site found at 934 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 298
Illegal NheI site found at 75
Illegal NheI site found at 1278
Illegal PstI site found at 934 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 298
Illegal BglII site found at 187
Illegal BglII site found at 1216
Illegal BamHI site found at 386 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 298
Illegal PstI site found at 934 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 298
Illegal PstI site found at 934
Illegal AgeI site found at 835 - 1000COMPATIBLE WITH RFC[1000]
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 CPS-1. It encodes a protein which is CPS-1 fused with GST tag. The fusion protein is about 59.2 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of CPS-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 CPS-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.
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