Part:BBa_K2933155

His+Linker f+VIM-66

This part encodes the fusion protein of His tag and VIM-66 to promote the expression and purification of target protein(VIM-66).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 51
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 824
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE 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 VIM-66. It encodes a protein which is VIM-66 fused with His tag. The fusion protein is about 28.3 kD. It is convenient for us to purify our target protein.

Molecular cloning

We insert VIM-66 gene into the standard vector then transfer it into E.coli.

Figure 1. Left: The result of PCR, Right:The result of double enzyme digestion verification.LaneM,Marker, Lane1, the plasmid with VIM-66, Lane2, after double enzyme verification

References

1. Yoshihiro Yamaguchi. Wanchun Jin. Kazuyo Matsunaga. Crystallographic investigation of the inhibition mode of a VIM-2 metallo-beta-lactamase from Pseudomonas aeruginosa by a mercaptocarboxylate inhibitor. J. Med. Chem.200750266647-6653

2. Biochemical, Mechanistic, and Spectroscopic Characterizationof Metallo-β-lactamase VIM‑2[J]. Biochemistry, 2014, 53(46):7321-7331.

3. Christopeit T , Carlsen T J , Helland R , et al. Discovery of novel inhibitor scaffolds against the metallo-β-lactamase VIM-2 by SPR based fragment screening[J]. Journal of Medicinal Chemistry, 2015:151017114758002.

4. Christopeit T , Yang K W , Yang S K , et al. The structure of the metallo-β-lactamase VIM-2 in complex with a triazolylthioacetamide inhibitor[J]. 2016.