Difference between revisions of "Part:BBa K3147005"

Revision as of 10:30, 18 October 2019

TEV protease with TEV-cleavable maltose binding protein

I: Part BBa_K3147005 (MBP-TEVcs-TEV) function

The 2019 Montpellier iGEM team made this construction in order to compare the basal activity of the TEV protease (BBa_ K1319004) with the same protease fused to a VHH nanobody. This construction was used as a control within the framework of the KARMA project. This construction produces an MBP fused to a TEV protease separated by a TEV cutting site (BBa_ J18918). MBP increases the solubility of the fusion protein [1], preventing the aggregation of the protein of interest. This stabilizes the expression and the sequence of the produced MBP does not have a signal peptide, which allows the protein to remain in the cytosol. The TEV cutting site allows self-cleavage of MBP from TEV once the protein is produced [2]. An affinity 7His-tag was added to N-terminus of the TEV protease to facilitate protein production and cleavage validation via Western blot and protein purification if desired.

II. Proof of function

The experimental approach to test protease activity is to compare the fluorescence restoration rate of sfGFP comparing MBP-TEV and MBP-TEV fused to a VHH. 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 TEV cleavage site used is the classic ENLYFQ site, MBP stabilizes the expression and increases the solubility of the fusion protein. The TEV protease used is optimized for expression in E. coli. The sequence contains an cleavage mutation "S219V anti-self-cleavage".

In order to carry out this experiment we used e.coli bacteria of the NEB10B strain. Fluorescence data are obtained by a Plate Reader at 30°C and 37°C. The protease is expressed by inducing the TET promoter with 50ng/mL of aTc (anhydrotetracycline). In this experiment, basal controls of maximum and minimum fluorescence of reporter genes were used.

Reference

[1] Raran-Kurussi, Sreejith, et David S. Waugh. 2012. « The Ability to Enhance the Solubility of Its Fusion Partners Is an Intrinsic Property of Maltose-Binding Protein but Their Folding Is Either Spontaneous or Chaperone-Mediated » éd. Bostjan Kobe. PLoS ONE 7(11): e49589.

[2] Shih, Y.-P. 2005. « Self-Cleavage of Fusion Protein in Vivo Using TEV Protease to Yield Native Protein ». Protein Science 14(4): 936‑41.

Sequence and Features