Difference between revisions of "Part:BBa K2933103"
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===Usage and Biology=== | ===Usage and Biology=== | ||
| − | This composite part is made up with 5 basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein ElblaII. It encodes a protein which is | + | This composite part is made up with 5 basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein ElblaII. It encodes a protein which is ElblaII fused with His-Sumo tag. The fusion protein is about 39.5kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of ElblaII 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. |
===Molecular cloning=== | ===Molecular cloning=== | ||
Revision as of 14:49, 28 September 2019
His+Linker a+Sumo+Linker b+ElBlaII
This part encodes the fusion protein of His-Sumo tag and ElBla to promote the expression and purification of target protein(ElBlaII).
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
Illegal AgeI site found at 839 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This composite part is made up with 5 basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein ElblaII. It encodes a protein which is ElblaII fused with His-Sumo tag. The fusion protein is about 39.5kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of ElblaII 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.
Molecular cloning
First, we used the vector pET-28b sumo 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 ElblaII. 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] Girlich D, Poirel L, Nordmann P, Diversity of naturally occurring Ambler class B metallo-β-lactamases in Erythrobacter spp. The Journal of Antimicrobial Chemotherapy [31 Jul 2012, 67(11):2661-2664]