Part:BBa_K2933150
His+Linker f+GIM-2
This part encodes the fusion protein of His tag and GIM-2 to promote the expression and purification of target protein(GIM-2).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 817
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 51
Illegal PstI site found at 817 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 817
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 817
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This composite part is made up with three basic parts, the HiS tag, the thrombin restriction site and our target protein GIM-2. It encodes a protein which is GIM-2 fused with His tag. The fusion protein is about 27.4 kD. It is convenient for us to purify our target protein.
Molecular cloning
First, we used the vector pET28a to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.
Figure 1. The result of PCR
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| None |