Difference between revisions of "Part:BBa K2549041"

Revision as of 13:00, 17 October 2018

NLS-TEVp

This part is one of the downstream elements of our amplifier. It is constructed by fusing NLS (Part:BBa_K2549054) and TEV protease (Part:BBa_K2549013), from N terminal to C terminal. NLS is a short nuclear location sequence from SV40 large T antigen. TEVp is a mutant TEV (tobacco etch virus) protease whose autoactivation is removed, thus performing more efficient cleavage^[1]. When coexpressed with VP64-dNLS-ZF21.16 (Part:BBa_K2549039) in the same cell, it can destroy the nuclear localization sequence and prevent the transcription activator from inducing the gene expression. Similarly, the cleavage can be conducted with the KRAB-dNLS-ZF21.16 (Part:BBa_K2549040), thus keeping the repressor from switching off the gene expression.

Biology

TEV protease-based transcription regulation or proteolysis-only activity

TEV protease is widely used in synthetic biology for its high cleavage specificity (targetting amino acids sequence ENLYFQG/S between QG or QS)^[2]. Rossner MJ et al have demonstrated the TEV activity-dependent activation of several reporters^[3]. In our system, we set the TEV protease specific cleavage site into the spacer region of the nuclear localization sequence^[4], which is the core design of the TEV protease-based complex logic gates.

Rossner MJ et al stated:TEV protease activity can be monitored with either inactivated transcription factors (‘transcription-coupled’ reporters) or inactivated reporter proteins (‘proteolysis-only’ reporters). We designed a ‘transcription-coupled’ TEV reporter at the membrane (TM-GV) by fusing a transcription factor composed of a yeast Gal4 DNA-binding domain and the herpes simplex VP16 transactivation domain (GV) to an hemagglutinin (HA)-tagged transmembrane region from the human PDGF-a receptor via a TEV-protease cleavage site ENLYFQ'G . After TEV protease cleavage, GV can translocate into the nucleus and induce reporter gene expression via five clustered Gal4-responsive cis elements. We constructed membrane localized ‘proteolysis-only’ reporters containing a luciferase moiety. We flanked GV N- and C-terminally by tevS sites and ERT2 domains (GV-2ER) thereby trapping the GV transcription factor reporter unit in the cytoplasm.

For more details about TEV protease, please refer to our TEVp (Part:BBa_K2549013).

Characterization

It works as we designed.

TEVp-based IMPLY gate. A degradable EGFP (d2EGFP) is linked downstream the promoter to indicate the expression level of it. DBD, DNA binding domain which is zinc finger in our assay. AD or SD, activating- or silencing-form transcriptional domain. RE, responsive elements. RFI, relative fluorescence intensity.

Flow cytometry results indicate that when KRAB-dNLS-ZF21.16 exists while TEVp doesn't exist that the expression level of d2EGFP is repressed. When TEVp and KRAB-dNLS-ZF21.16 coexist, TEVp cleaves dNLS and prevents KRAB from entering into the nucleus. Thus the repression is not severe as when KRAB-dNLS-ZF21.16 is present while TEVp is absent.

Sequence and Features

References

1. ↑ Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Kapust RB, Tözsér J, Fox JD, ..., Copeland TD, Waugh DS. Protein Eng, 2001 Dec;14(12):993-1000 PMID: 11809930
2. ↑ Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase. Parks TD, Leuther KK, Howard ED, Johnston SA, Dougherty WG. Anal Biochem, 1994 Feb;216(2):413-7 PMID: 8179197; DOI: 10.1006/abio.1994.1060
3. ↑ 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
4. ↑ Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Robbins J, Dilworth SM, Laskey RA, Dingwall C. Cell, 1991 Feb;64(3):615-23 PMID: 1991323