Part:BBa_K2549042

NLS-TEVpN-CfaN

This part is one of the downstream elements of our amplifier. It is constructed by fusing NLS (Part:BBa_K2549054), TEVpN (Part:BBa_K2549014) and CfaN (Part:BBa_K2549009), from N terminal to C terminal. NLS is a short nuclear location sequence from SV40 large T antigen. TEVpN is the N-terminal fragment of TEV protease. TEV protease is one of the best-characterized enzymes of the viral proteases which have more stringent sequence specificity. CfaN is the N-terminal fragment of Cfa which is a consensus sequence from an alignment of 73 naturally occurring DnaE inteins that are predicted to have fast splicing rates^[1]. When coexpressed with CfaC-TEVpC (Part:BBa_K2549043) in the same cell, two fusion proteins can form and the site-specific cleavage can be realized.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 337

Biology

Please visit http://2018.igem.org/Team:Fudan/Results and http://2018.igem.org/Team:Fudan/Measurement to check how we use this.

split TEV protease

MJ Rossner et al have demonstrated a biological assay termed split TEV. They engineered inactive fragments of the NIa protease from the tobacco etch virus (TEV protease) that regain activity only when coexpressed as fusion constructs with interacting proteins^[2].

MJ Rossner et al demonstrated:In a systematic screen for transcomplementation-competent N and C-terminal TEV protease fragments (referred to as N-TEV and C-TEV), we found several fragment pairs that showed substantial recapitulation of activity upon specific interaction of model-protein dimerization domains to which they had been fused. We obtained the best results using the two fragment pairs N-TEV(1–118)/C-TEV(119–242) and N-TEV(1–70)/C-TEV(61–242). Both pairs displayed B30–40% activity compared to the full length TEV protease, a three- to fourfold increase over the control assay.We used the N-TEV(1–118)/C-TEV(119–242) pair for all subsequent experiments.

Thus in our split TEV protease assay, we chose site between 118 amino acid and 119 amino acid of TEV protease as the split site.

intein-based protein splicing

Schematic below provides a mechanism of how the intein function^[3].

The mechanism of protein splicing involving inteins. In this scheme, the N-extein is shown in red, the intein in black, and the C-extein in blue. X represents either an oxygen or sulfur atom.

For more details about intein-based protein splicing, please refer to our CfaN (Part:BBa_K2549009) and CfaC (Part:BBa_K2549010).

References

↑ Design of a Split Intein with Exceptional Protein Splicing Activity. Stevens AJ, Brown ZZ, Shah NH, ..., Cowburn D, Muir TW. J Am Chem Soc, 2016 Feb;138(7):2162-5 PMID: 26854538; DOI: 10.1021/jacs.5b13528
↑ Monitoring regulated protein-protein interactions using split TEV. Wehr MC, Laage R, Bolz U, ..., Nave KA, Rossner MJ. Nat Methods, 2006 Dec;3(12):985-93 PMID: 17072307; DOI: 10.1038/nmeth967
↑ https://en.wikipedia.org/wiki/Intein

[1] Design of a Split Intein with Exceptional Protein Splicing Activity. Stevens AJ, Brown ZZ, Shah NH, ..., Cowburn D, Muir TW. J Am Chem Soc, 2016 Feb;138(7):2162-5 PMID: 26854538; DOI: 10.1021/jacs.5b13528

[2] Monitoring regulated protein-protein interactions using split TEV. Wehr MC, Laage R, Bolz U, ..., Nave KA, Rossner MJ. Nat Methods, 2006 Dec;3(12):985-93 PMID: 17072307; DOI: 10.1038/nmeth967

[3] ttps://en.wikipedia.org/wiki/Intein

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