Difference between revisions of "Part:BBa K4241019"
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Purification scheme:<br/> | Purification scheme:<br/> | ||
| − | 1. Express fusion protein and sonicate cells | + | 1. Express fusion protein and sonicate cells <br/> |
| − | 2. Centrifuge and remove supernatent. The fusion protein aggregates will form and fall to the bottom. | + | 2. Centrifuge and remove supernatent. The fusion protein aggregates will form and fall to the bottom. <br/> |
| − | 3. Treat with DTT to cleave the protein of interest from the aggregates | + | 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=== | ||
Revision as of 13:26, 9 October 2022
RFP_Mxe_GyrA_Intein_PT_ELK16
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1482
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1305
Illegal NgoMIV site found at 1320
Illegal AgeI site found at 655
Illegal AgeI site found at 767 - 1000COMPATIBLE WITH RFC[1000]
Overview
This is composite part is RFP 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.
Fig. 1. Left: Sonicated bacteria RFP-Intein-ELK16 system. Right: Sonicated bacteria 6xHis-SUMO-RFP.
Fig. 2. SDS PAGE results depicting cleaved RFP after overnight DTT cleavage of RFP-intein fusion.
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.
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.
Parts that comprise this composite component
T7pCONS-TIR-2 - Part:BBa_K4241015
highly engineered mutant of red fluorescent protein from Discosoma striata (coral) - Part:BBa_E1010
Mxe_GyrA_Intein with PT linker and ELK16 - Part:BBa_K4241018
Double stop codon - Part:BBa_K4241018
double terminator (B0010-B0012) - Part:BBa_B0015