Difference between revisions of "Part:BBa K4241018"
 
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<br/>
 
<br/>
 
Purification scheme:<br/>
 
Purification scheme:<br/>
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1. Express fusion protein and sonicate cells
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1. Express fusion protein and sonicate cells <br/>
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2. Centrifuge and remove supernatent. The fusion protein aggregates will form and fall to the bottom.
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2. Centrifuge and remove supernatent. The fusion protein aggregates will form and fall to the bottom. <br/>
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3. Treat with DTT to cleave the protein of interest from the aggregates
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3. Treat with DTT to cleave the protein of interest from the aggregates <br/>
−
4. Centrifuge and remove supernatent. The pure, protein will be solubized whereas the uncleaved protein and insoluble aggregates will remain at the bottom.
+
4. Centrifuge and remove supernatent. The pure, protein will be solubized whereas the uncleaved protein and insoluble aggregates will remain at the bottom. <br/>
  
 
===Results===
 
===Results===
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Fig. 2. SDS PAGE results depicting cleaved RFP after overnight DTT cleavage of RFP-intein fusion.
 
Fig. 2. SDS PAGE results depicting cleaved RFP after overnight DTT cleavage of RFP-intein fusion.
 
<br/>
 
<br/>
 +
 +
===Improvement upon an existing part===
 +
This part is an improvment upon the following part:<br/>
 +
Mxe GryA intein-PT-linker-ELK16 for peptide production - BBa_K3815000 <br/>
 
<br/>
 
<br/>
−
The RFP was precipitated in 2.5% acetic acid to release the RFP from the intein fusion. It was then resolubized in with urea and high salt solution. The ELK16 remains in the insoluble fraction.
+
Improvements:<br/>
−
[[File:BBaK4241018-3.jpeg|center]]
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The intein sequence has been corrected <br/>
−
Fig. 3. RFP is denatured with acetic acid (as shown by the loss of color), but could be refolded in 8M Urea to regain RFP red colour rapidly to be back to natural folding.
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The part is annotated in the correct direction
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===References===
 +
Wang, M., Zheng, K., Lin, J., Huang, M., Ma, Y., Li, S., Luo, X., &amp; Wang, J. (2018). Rapid and efficient production of cecropin a antibacterial peptide in escherichia coli by fusion with a self-aggregating protein. BMC Biotechnology, 18(1). https://doi.org/10.1186/s12896-018-0473-7
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 16:52, 9 October 2022


Mxe_GyrA_Intein with PT linker and ELK16


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 676
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 499
    Illegal NgoMIV site found at 514
  • 1000
    COMPATIBLE WITH RFC[1000]

Overview

This is part is to be fused to a target protein of interest for 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.

Results

After sonication, the protein is released from the cells. In cells expressing intein-ELK16, the RFP-tagged fusion protein remains in an aggregated and insoluble fraction.

BBaK4241018-1.jpeg

Fig. 1. Left: Sonicated bacteria RFP-Intein-ELK16 system. Right: Sonicated bacteria 6xHis-SUMO-RFP.

BBaK4241018-2.jpeg

Fig. 2. SDS PAGE results depicting cleaved RFP after overnight DTT cleavage of RFP-intein fusion.

Improvement upon an existing part

This part is an improvment upon the following part:
Mxe GryA intein-PT-linker-ELK16 for peptide production - BBa_K3815000

Improvements:
The intein sequence has been corrected
The part is annotated in the correct direction

References

Wang, M., Zheng, K., Lin, J., Huang, M., Ma, Y., Li, S., Luo, X., & Wang, J. (2018). Rapid and efficient production of cecropin a antibacterial peptide in escherichia coli by fusion with a self-aggregating protein. BMC Biotechnology, 18(1). https://doi.org/10.1186/s12896-018-0473-7