Difference between revisions of "Part:BBa K3147011"
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Figure 2: MBP-TEVPE10-VHH reporter gene in its backbone pOUT18. | Figure 2: MBP-TEVPE10-VHH reporter gene in its backbone pOUT18. | ||
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Figure 3 : Comparison of the activity of VHH-TEVPH21 to the VHH-TEV WT by measuring the fluorescence of a reporter that needs to be cutted from a proteolysis tag | Figure 3 : Comparison of the activity of VHH-TEVPH21 to the VHH-TEV WT by measuring the fluorescence of a reporter that needs to be cutted from a proteolysis tag | ||
Revision as of 12:11, 14 October 2019
Mutant TEV (PH21) protease fused to anti-sfGFP VHH with TEV-cleavable maltose binding protein
I : parts BBa_K3147011 (MBP-TEVcs-TEV) function :
The Montpellier 2019 team made this construction in order to be able to compare the basal activity of the TEV PH21 protease with the same fused protease to a nanobody type VHH. 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 modified TEV cut-off site 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 signal peptide which allows to keep the protein in the cytosol. The TEV cutting site allows to separate MBP from TEV once the protein is produced, indeed the TEV protease is capable of self-cleavage to remove MBP [2]. An affinity tag 6 histidine is added in C-ter of the VHH.
Figure 1 : Construct Design: MBP-TEVcs-TEVPH21-VHH with modified TEV cutting site. The objective of the construction is to be able to compare the activity of a TEV protease alone against a TEV protease fused to an anti sfGFP VHH.
II. Proof of function
The construction was cloned by Gibson Assembly in a backbone pOUT18 under the control of a TET ON promoter, in order to control its expression. The experimental approach to test protease activity is to compare the fluorescence restoration rate of sfGFP against MBP-TEV-VHH and MBP-TEVPH21-VHH. In this experiment, basal controls of maximum and minimum fluorescence of reporter genes were used. Fluorescence data are obtained by Plate Reader. The protease is expressed by inducing the TET promoter with 50ng/mL of aTc (anhydrotetracycline).
Figure 2: MBP-TEVPE10-VHH reporter gene in its backbone pOUT18.
Figure 3 : Comparison of the activity of VHH-TEVPH21 to the VHH-TEV WT by measuring the fluorescence of a reporter that needs to be cutted from a proteolysis tag
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 2377
Illegal SapI.rc site found at 1868