Part:BBa_K2933020
subclass B1 metallo-beta-lactamase IMP-71, codon optimized in E. coli
This part encodes a protein called IMP-71, which is a metallo-beta-lactamase of subclass B1.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 724
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 724
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 724
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 724
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Bacteria with IMP-type enzymes have spread through out the world, and the IMP group now has more than 50 variants
These enzymes possess a broad substrate specificity and a high affinity for cephalosporins and carbapenems but a low activity toward temocillin
References
[1]Purification, crystallization and preliminary X-ray analysis of IMP-18, a class B carbapenemase from Pseudomonas aeruginosa.Furuyama T, Ishii Y, Ohya N, Tateda K, Hanson ND, Shimizu-Ibuka A.Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013 Dec;69(Pt 12):1397-400.
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 IMP-71. 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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