Part:BBa_K3147009
TEV protease fused to anti-sfGFP VHH with TEV-cleavable maltose binding protein
The Montpellier 2019 team made this structure in order to be able to compare the basal activity of the TEV protease (BBa_ K1319004) with the same fused protease to a VHH-type antibody This construction produces an MBP fused to a TEV protease attached to a specific VHH of the sfGFP . Between the MBP and the protease a TEV cutting site (BBa_ J18918) is added. MBP increases the solubility of the fusion protein [1] preventing the aggregation of the protein of interest, this stabilizes the expression, the sequence of the produced MBP does not have a peptide signal which allows to keep the protein in the cytosol. The TEV cutting site allows the separation of MBP from TEV once the protein is produced, in fact the TEV protease is capable of self-cleavage to remove MBP [2]. An affinity tag 7 histidine is added in N-ter of the TEV protease that can allow us to see the separation activity of the TEV by a Western-Blot.
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Figure 1 : Construct Design: MBP-TEVcs-TEV-VHH with TEV cutting site. The objective of the construction is to be able to compare the activity of a TEV protease alone versus a TEV protease fused to a VHH.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 77
Illegal BsaI.rc site found at 2398
Illegal SapI.rc site found at 1541
Illegal SapI.rc site found at 1889
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