Part:BBa_K4041020
Expression Cassette for pET-LO-SUMO-AMP1-Tachy 4303
HKIS Improvement
A AI model machine learning has been ran to improve the cytotoxicity of the peptide and generated several mutant candidates (930, 4303, 5305.) This is an improvement to the Part:BBa_K1835501
This is for the surface display of the protein SUMO-Tachyplesin or Tachy variant to be cleaved by the Yafl-Ulp1-protein partner into SUMO and tachy into the cleavage solution outside of the cell. This will release a 2kDa AMP into the solution and thus would be purified. Which in turn purifies the protein/peptide of choice without the use of tags but only centrifugation. This method allows easy centrifugation to purify protein in a relatively pure output.
Figure 2, Illustration of Cleavage system (brown Yafl Ulp1, Red fusion protein)
Source: https://amb-express.springeropen.com/articles/10.1186/s13568-020-00999-4
This system does not work well with system that does cannot bear the toxicity, like the BL21 (DE3) -gold cells, which has a high mRNA stability.
Figure 2, showing the expression of Yafl from our own test, pET-LO-SUMO and pET-LO-SUMO-tachyplesin system.
From the picture, it is suggested to use lysis resistant strains and other strains with high toxicity tolerance for the IPTG induction of the peptide, preferably in high enrichment broth including terrific broth or magic media.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 1189
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 2033
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1147
Illegal BglII site found at 1820 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 1189
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 1189
Illegal AgeI site found at 1630 - 1000COMPATIBLE WITH RFC[1000]
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