Part:BBa_K2933135

His+Linker a+Sumo+Linker b+VIM-66

This part encodes the fusion protein of His tag, sumo tag and VIM-66 to promote the expression and purification of target protein(NDM-23).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 256
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 256
  Illegal NheI site found at 33
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 256
  Illegal BglII site found at 145
  Illegal BglII site found at 1093
  Illegal BamHI site found at 344
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 256
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 256
  
• 1000
  COMPATIBLE 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 VIM-66. It encodes a protein which is VIM-66 fused with His-Sumo tag. The fusion protein is about 40.3kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of VIM-66 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

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

Exploration of expression condition

Figure 2. The result of SDS-PAGE.
Lane1, uninduced VIM-66 with His tag(BBa_K2933155)(28.3kD).
Lane2, 37°C induced VIM-66 with His tag(28.3kD)
with 0.5mM IPTG.

Expression and purification

Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with kanamycin(50 μg/mL final concentration) in 37℃ overnight.

Massive expressing:
After taking samples, we transfer them into 1L LB medium and add antibiotic to 50 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours). Induce the culture to express protein by adding 0.5 mM IPTG (isopropylthiogalactoside, MW 238 g/mol) or ~0.1 gram per 1.5 liter flask. Put the liter flasks in 16°C shaking incubator for 16h. Centrifuge your bacteria in 500 mL bottles in the 4°C rotor at 4,000 rpm for 20 mins. Do this in batches until all your culture is spun down saving the cell pastes each time.

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.