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From 2014.igem.org

Contents 1 Introduction 1.1 Controlling Inteins 1.2 The As LOV2 domain 1.3 S11 split inteins 2 Cloning and Methods 3 Results 3.1 Screening 3.2 Brilliant Data … 4 Discussion 5 Outlook 5.1 References

Introduction

Protein splicing allows for the introduction of major changes to a proteins sequence and structure even after translation. Although this mechanism alone offers a huge amount of possible applications highlighted in our toolbox, it would be advantageous to have control over the time and location of the splicing reaction especially for in vivo applications. However, the fact that inteins are attached to the protein they splice off from makes induction in whatever way rather difficult.

Controlling Inteins

Different ways exist for controlling the intein splicing reaction. In the literature this is mostly referred to as conditional trans splicing (CTS) or conditional protein splicing (CPS). Probably the longest known method of inducing protein splicing is to chemically control the activity of inteins by changing the pH or redox state of their environment. This has been achieved for a multitude of inteins such as Ssp DnaB [#References|[5]] or Mtu recA [10 . Yet the major drawback is obvious:These methods are only effectively applicable in vitro and usually depend on complexly purified reagents.

Within the past decades, more advanced systems were discovered and designed involving the binding of different ligands or the fusion of inteins to various protein domains Ways of controlling inteins (Conditional Trans-Splicing!) -> splicing reaction, chemically: pH, redox-state -> ligand or light dependant (PhyB/PIF3, Calmodulin?) protein domain “assembling”/complementation? -> This only works for less active inteins, because otherwise they are more likely to attach than the -> new way needed

We therefore came up with a new method to inhibit an inteins function light-dependently: LOV2 domain photocaging.

The As LOV2 domain

As LOV2 is the second Light-Oxygen-Voltage domain from the Avena sativa phototropin 1. In its host organism, the common oat, phototropin 1 is a blue-light receptor involved in the response of growth to environmental light conditions and may be responsible for the opening of stomata and the movement of chloroplasts [4]. LOV2 mainly consists of a flavin mononucleotide (FNM) -binding core containing a FNM as chromophore, as well as a helical structure on the C-terminus, the J-alpha helix. Upon excitation with light, a cysteine within the core covalently binds the activated FMN, which induces a conformational change resulting in the protrusion of the formerly concealed J-alpha helix.

This light dependent change in conformation can be used to control cellular processes via light induction, a method for which two strategies exist: It has been shown, that proteins or protein domains which are fused C-terminally to the LOV2 are not able to bind to possible interaction partner due to sterical hindrance. Only after the conformational change initiated by illumination the J alpha helix is exposed, and so is the attached protein domain and its catalytic core. [6]. Instead of being fused to the very end, small amino acid sequences can be introduced and therefore hidden within the J alpha helix. Usually, this offers a better light-state to dark-state (signal to noise) ratio. This approach, also termed photocaging, has already been used to control degradation or nuclear accumulation of proteins [7][8].

It has been suggested that the length of the caged peptide itself determines the success of the photo caging [9. Thus, we screened the literature and discovered a set of 6 amino acid long C – inteins.

In the context of cell biology and engineering signalling pathways

Normal use -> allosteric? effect/spatially blocking active centre (would be second way of controlling as seen above…) -> but …

photocaging offers better way Definition -> small peptide in Jalpha Helix

Problem: small Inteins needed

S11 split inteins

WHY the hell? Where from, where split, name reference!

Cloning and Methods

Results

During the planning phase two preparatory experiments were run to test the conditions for light induction: Please visit conditions testing to read more about the procedures and results.

The cloning was performed on the basis of our two-gate assembly part. However, due to the need for fast cloning, all constructs were made CPEC. For each split fluorescent protein, the constructs were assembled directly into pSBX1K3:

• split N-protein and N-intein
• split C-intein and C-protein
• split non-splicing N-protein and N-intein
• split non-splicing C-intein and C-protein
• LOV domain with caged C-Intein position 6
• LOV domain with caged C-intein position 12
• (only for mRFP: LOV domain with BsaI cloning site for further customized caging positions)
• mutated positive controls

For split sfGFP all N splicing-partner and control constructs were taken from the split fluorescent protein assay.

Cloning scheme:

plasmids --- PCR ---> PCR products --- CPEC ---> constructs

Screening

Brilliant Data …

Discussion

Outlook

reference to Cas9 + possible applications (Philipps idea)

References

[1] Aranko, A. S., Oeemig, J. S., Kajander, T. & Iwaï, H. Intermolecular domain swapping induces intein-mediated protein alternative splicing. Nat. Chem. Biol. 9, 616–22 (2013).

[2] Strickland, D. et al. TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat. Methods 9, 379–84 (2012).

[3] Lin, Y. et al. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins. PLoS One 8, e59516 (2013).

[4] Deblasio, S. L., Luesse, D. L. & Hangarter, R. P. A Plant-Specific Protein Essential for Blue-Light-Induced Chloroplast Movements 1. 139, 101–114 (2005).

[5] Lu, W. et al. Split intein facilitated tag affinity purification for recombinant proteins with controllable tag removal by inducible auto-cleavage. J. Chromatogr. A 1218, 2553–60 (2011).

[6] Wu, Yi I., et al. A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461.7260 (2009), 104-108.

[7] Renicke, Christian, et al. A LOV2 domain-based optogenetic tool to control protein degradation and cellular function. Chemistry & biology 20.4 (2013), 619-626.

[8] Niopek, Dominik, et al. Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells. Nature communications 5 (2014).

[9] Yi, Jason J., et al. Manipulation of Endogenous Kinase Activity in Living Cells using Photoswitchable Inhibitory Peptides. ACS Synthetic Biology (2014).

[10] Wood, D. W., Wu, W., Belfort, G., Derbyshire, V. & Belfort, M. A genetic system yields self-cleaving inteins for bioseparations. 889–892 (2002).