Part:BBa_K2942704:Design

Carboxypeptidase

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1304
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 610
  Illegal NgoMIV site found at 808
  Illegal NgoMIV site found at 1237
  Illegal AgeI site found at 1318
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 160
  

Carboxypeptidase(CPG2)

Carboxypeptidase is a kind of peptidase that specifically degrades and releases free amino acids from the C end of the peptide chain. And the ligand we use in our project is G2 type which could cleave C-terminal Glu under N-acetyl-L-aspartyl-L-glutamate structure. CPG2 is a zinc-dependent dimeric protein with no mammalian analogue [1], thus in our project we carried out human codon optimization on the original CPG2 and added flag tag for labeling to increase protein expression and make detection easier. CPG2 rapidly hydrolyzes extracellular MTX to its non-toxic metabolites, called 2, 4-diamino-N10-methypteroic acid and glutamic acid [2], so it can be used for the treatment of elevated plasma concentrations of MTX. The principle of this reaction shows in the picture as follows.

Mohammad A et al. Glucarpidase (Voraxaze), a Carboxypeptidase Enzyme for Methotrexate Toxicity. P T. 2013 Dec; 38(12): 732, 741-744.

This part was mainly used for the construction of composite part. We artificially synthesized the sequence, and then we design the primers (see details in the composite part BBa_2942706) to gain and recycle the PCR product (Fig.1) for the multiple fragment homologous recombination next. Its characterization can be seen in the composite part BBa_2942706 in details.

Fig.1 PCR result. We followed the protocol of NEB Q5® High-Fidelity 2X Master Mix to perform the PCR, and the PCR product was identified by 1% agarose gel electrophoresis.

[1] S. Rowsell, R.A. Pauptit, A.D. Tucker, R.G. Melton, D.M. Blow, P. BrickCrystal structure of carboxypeptidase G 2, a bacterial enzyme with applications in cancer therapy Structure, 5 (3) (1997), pp. 337-347

[2] D. Mitrovic, D. Touw, W. TissingTreatment of high dose methotrexate toxicity with Glucarpidase. J. Clin. Toxicol., 6 (293) (2016), pp. 0495-2161

Design Notes

We put this sequence at the downstream of the riboswitch to control its expression and we should make the number of the sequence base multiple of three to avoiding frameshift.

Source

Artificially Synthesized

References

[1] S. Rowsell, R.A. Pauptit, A.D. Tucker, R.G. Melton, D.M. Blow, P. BrickCrystal structure of carboxypeptidase G 2, a bacterial enzyme with applications in cancer therapy Structure, 5 (3) (1997), pp. 337-347

[2] D. Mitrovic, D. Touw, W. TissingTreatment of high dose methotrexate toxicity with Glucarpidase. J. Clin. Toxicol., 6 (293) (2016), pp. 0495-2161