Part:BBa_K4794008
T7-His tag-SUMO tag-intein-defensin-like peptide 4(DLP4)
The construct we designed contains T7 promoter, SUMO-tag, 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].
SUMO-tag: It is the most frequently used as N-end fusion sequence to increase the expression and solubility of the desired recombinant protein. Attachment can be removed by SUMO-specific isopeptidases just as UCHs and UBPs remove ubiquitin from substrate proteins.
6xHis Tag: It is one of the simplest and most widely used purification tags and commonly used in the production of recombinant proteins. These residues readily coordinate with transition metal ions immobilized on beads or a resin for purification.
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 BglII site found at 158
Illegal XhoI site found at 753 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |