Difference between revisions of "Part:BBa K4241019"

Revision as of 16:58, 9 October 2022

RFP_Mxe_GyrA_Intein_PT_ELK16

Overview

This is composite part is RFP 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.

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

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