Part:BBa_K3794004:Design

ChABC CDS

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2754
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

This part's DNA sequence was derived from PDB 1HN0, and had its signal peptide sequence (amino acids 1-24) removed. A 6xHis tag + TEV site with upstream start codon were incorporated to allow for transcriptional initiation and subsequent protein purification via a Ni-NTA column.

Due to ChABC's thermal instability, a series of 8 point mutations derived from "Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability" (Hettiaratchi et al., 2020) were incorporated into the protein sequence.

These mutations are as follows:

• K194E
• A228K
• S274P
• N288D
• S343N
• K654D
• R670T
• Q781E

The DNA sequenced derived from this protein sequence was codon optimised for expression in E.coli K12.

A stop codon was added at the 3' end of the ChABC CDS to allow for translational termination during protein synthesis.

Source

Synthesised from IDT. Protein sequence obtained from PDB: 1HN0 and signal peptide removed. Mutations obtained from "Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability" (Hettiaratchi et al., 2020)

References

Hettiaratchi, Marian & O'Meara, Matthew & O'Meara, Teresa & Pickering, Andrew & Letko-Khait, Nitzan & Shoichet, Molly. (2020). Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability. Science Advances. 6. eabc6378. 10.1126/sciadv.abc6378.