Part:BBa_K4794010
T7-defensin-like peptide 4(DLP4)-intein-PT linker-ELK16
The construct we designed contains T7 promoter, ELK16 system, Polyhistidine-tag (6-His tag), intein and defensin-like peptide 4(DLP4), a antimicrobial peptide orginated from black soldier fly[BBa_K4794000]. Moreover, SacI and KpnI, two restriction sites are designed on both ends of DLP4, so that the DLP4 can be replaced with another AMPs easily[BBa_K4794001, BBa_K4794002, BBa_K4794003, BBa_K4794004, BBa_K4794005 and BBa_K4794006].
Our previous design[BBa_K4794008] has a problem of low solubilty. The results of SDS-PAGE showed that most of expressed proteins for DLP1-3, were found in the pellet instead of supernatant, which greatly reduced the yield of AMPs collected for the purification and thus the antimicrobial test. Also, the result show that intein self-cleavage system works but the efficiency can be increased with further fine tuning of the cleavage environment or even with alternate design of cleavage system.
In order to increase the efficiency of AMPs purification and self-cleavage, we have designed this new composite part in which SUMO tag and His tag are replaced by ELK-16 system to change the purification mechanism by changing the solubility of AMP from insoluble to soluble form. The ELK-16 system includes ELK16, Mxe GyrA intein, and PT linker. The protein expressed first is insoluble, but by adding DTT, the AMPs can be removed from intern and become soluble and functional. This is a easy way to purify target protein with intein system.
This system would be applied for future experiments (2nd engineering cycle). If this new system works, we can collect higher concentration of AMPs for antimicrobial test to achieve our final goals – finding an alternative of antibiotics and make antimicrobial black soldier fly ecoplaster.
ELK16 system: This system includes ELK16, Mxe GyrA intein, and PT linker. The protein expressed first is insoluble, but by adding DTT, the AMPs can be removed from intern and become soluble. This is a easy way to purify target protein with intein system.
Intein: Intein as a fusion protein to inhibit the action of AMPs inside E.coli before the purification and cleavage process. Intein-AMPs are inactivated antimicrobial peptide so that it will not kill E.coli during expression to ensure continuous expression of AMPs. These aggregates are easily and cheaply be purified by Ni-NTA resin (QIAGEN, Hilden, Germany) with the presence of His tag as well. Autocleavage excises itself when the recombinant protein is in pH 10 buffers.
T7 promoter: T7 promoter derived from the T7 bacteriophage. Allows high expression of proteins only when the T7 polymerase is present.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 62
Illegal XhoI site found at 310 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 778
- 1000COMPATIBLE WITH RFC[1000]
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