Difference between revisions of "Part:BBa K3815001"
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2.When this fused protein are produced,it self-assembles and precipitates.<br> | 2.When this fused protein are produced,it self-assembles and precipitates.<br> | ||
3.This aggregate is collected by centrifugation.<br> | 3.This aggregate is collected by centrifugation.<br> | ||
| − | 4.Adding DTT to this aggregate, the targeted protein peptides is cut out by the cleavage of intein | + | 4.Adding DTT to this aggregate, the targeted protein peptides is cut out by the cleavage of intein. |
<br> | <br> | ||
<br> | <br> | ||
Revision as of 15:56, 19 October 2021
CecropinA-Mxe GryA intein-PT-linker-ELK16
Usage and Biology
We used this part for purfication of antimicrobial peptide, CecropinA. This is derived from a moth. This is the antimicrobial peptide that can inhibit the growth of both gram-negative bacteria and gram-positive bacteria. In our experiment, we used it to kill the bacteria in the vase water.
This part is used for ELK16 system to purify the peptides. When this fused protein is produced, the part of ELK16 self-assembles and precipitates. After that, taking this aggregate and adding DTT to it, we cut the N terminal of Mxe GyrA intein. Then, we can get the targeted protein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 814
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 67
Illegal AgeI site found at 346 - 1000COMPATIBLE WITH RFC[1000]
Purification
Expression
- Cells were grown in 1000ml LB media at 37oC.
- when the OD exceeded 0.35, IPTG was added to induce the peptide expression.
Purification
1.E.coli which expressed this part were lysed with sonification.
2.When this fused protein are produced,it self-assembles and precipitates.
3.This aggregate is collected by centrifugation.
4.Adding DTT to this aggregate, the targeted protein peptides is cut out by the cleavage of intein.
From Fig1,