Part:BBa_K4241021

CeB_Mxe_GyrA_Intein_PT_ELK16

Assembly Compatibility:

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

  Illegal XhoI site found at 880
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 703
  Illegal NgoMIV site found at 718
  
• 1000
  COMPATIBLE WITH RFC[1000]

Overview

This is composite part is Cecropin B fused as a target protein of interest via inclusion expression. This part also features the ability for DTT mediated cleavage for downstream purification purposes. It is noted that this is a mutant version.
This is derived from the following publication: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173929/

Usage and Biology

ELK16 is the basis of a self-assembling peptide-fused protein expression system, SA-ELK16 system. ELK16 indues the formation of active fusion protein aggregates in E. coli, these aggregates are easiily and cheaply purified by centrifugation. The protein of interest can then be seperated by cleaving intein via DTT mediated cleavage. The summation of this allows for high purity, cost-effective purification scheme.

Purification scheme:
1. Express fusion protein and sonicate cells
2. Centrifuge and remove supernatent. The fusion protein aggregates will form and fall to the bottom.
3. Treat with DTT to cleave the protein of interest from the aggregates
4. Centrifuge and remove supernatent. The pure, protein will be solubized whereas the uncleaved protein and insoluble aggregates will remain at the bottom.

Parts that comprise this composite component

T7pCONS-TIR-2 - Part:BBa_K4241015
Cecropin B bacterial expression - Part:BBa_K4241020
Mxe_GyrA_Intein with PT linker and ELK16 - Part:BBa_K4241018
Double stop codon - Part:BBa_K4241018
double terminator (B0010-B0012) - Part:BBa_B0015