Difference between revisions of "Part:BBa K2933130"
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This composite part is made up with four basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein GIM-2. It encodes a protein which is GIM-2 fused with His-Sumo tag. The fusion protein is about 39.4kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of GIM-2 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. | This composite part is made up with four basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein GIM-2. It encodes a protein which is GIM-2 fused with His-Sumo tag. The fusion protein is about 39.4kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of GIM-2 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. | ||
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===Molecular cloning=== | ===Molecular cloning=== | ||
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'''Figure 1.''' Left: The result of PCR, Right:The result of double enzyme digestion verification | '''Figure 1.''' Left: The result of PCR, Right:The result of double enzyme digestion verification | ||
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==References== | ==References== | ||
Revision as of 13:42, 24 September 2019
His+Linker a+Sumo+Linker b+GIM-2
This part encodes the fusion protein of His tag, sumo tag and GIM-2 to promote the expression and purification of target protein(GIM-2).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 256
Illegal PstI site found at 1086 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 256
Illegal NheI site found at 33
Illegal PstI site found at 1086 - 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
Illegal PstI site found at 1086 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 256
Illegal PstI site found at 1086 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This composite part is made up with four basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein GIM-2. It encodes a protein which is GIM-2 fused with His-Sumo tag. The fusion protein is about 39.4kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of GIM-2 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
Figure 1. Left: The result of PCR, Right:The result of double enzyme digestion verification
References
[1]Skagseth S , Akhter S , Paulsen M H , et al. Metallo-β-lactamase inhibitors by bioisosteric replacement: Preparation, activity and binding[J]. European Journal of Medicinal Chemistry, 2017, 135:159-173.
[2]Wendel AF, MacKenzie CR. 2015. Characterization of a novel metallo-lactamase variant, GIM-2, from a clinical isolate of Enterobacter cloacae in Germany. Antimicrob Agents Chemother 59:1824 –1825.
[3]Borra P S , Samuelsen O , Spencer J , et al. Crystal Structures of Pseudomonas aeruginosa GIM-1: Active-Site Plasticity in Metallo-beta-Lactamases[J]. Antimicrobial Agents and Chemotherapy, 2013, 57(2):848-854.
[4]Susann S, Trine J C, Gro Elin K B, James S, Ørjan S, Hanna-Kirsti S. L. Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1. Antimicrobial Agents and Chemotherapy Jan 2016, 60 (2) 990-1002