"

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). The oldest 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 [1] or Mtu recA [2]. 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 designed involving the binding of different ligands or the fusion of inteins to various protein domains. For example Mootz et al. succeeded in inhibiting the splicing reaction of an synthetically engineered version of Ssp DnaE through the addition of rapamycin [3]. Similarly, the S. cerevisiae VMA intein has been conditionally activated by rapamycin induced complementation of FKBP and FRB protein domains. There have previously been attempts to light induce inteins with an analogous system in which the VMA intein halves are fused to the chromoproteins Phytochrome B (PhyB) and Phytochrome Associated Factor 3 (PIF3) [4]. Upon light induction at 660 λ nm PhyB and PIF3 assemble bringing the intein halves together to initiate splicing. Although most certainly a useful tool, these principle can only work for inteins with low affinity to each other and therefore with rather low reaction rates. When using faster inteins, the corresponding halves bind to each other with significantly higher affinity and thereby start the splicing reaction independently from the binding state of their coupled protein domains.

We therefore came up with a new method to inhibit an intein's function light-dependently using 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 [5]. 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 [6].

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. [7]. 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 [8][9].

As in LOV photocaging inhibition is not achieved by merely blocking an active centre but by completely hiding the whole binding partner we assessed the latter strategy as the most promising. Because of the C-terminal location of the J-alpha helix, the place that photocaging takes place in, an intein that is hidden within the LOV domain and splices off a fused protein without creating a significant scar needs to be a C-intein. It has been suggested that the length of the caged peptide itself determines the success of the photo caging [10]. Thus, we extensively screened the literature and discovered a set of only six amino acids long C–inteins.

S11 split inteins

Interestingly, there seem to exist only very few inteins with small C-terminal halves. The shortest and therefore most suitable were the S11 split inteins, engineered and thoroughly, but solely described by Lin et al. [3]. S11 inteins are engineered from mini-inteins, meaning that, unlike most other inteins, they do not have an internal homing endonuclease domain. Naturally, they are not trans-, but cis-splicing inteins and their crystal structure reveals twelve β-strands. To gain very small C-terminal split inteins, they were artificially separated between the 11th and 12th β-strand resulting in a 120-160&shortsp;aa N- and 6&shortsp;aa C-intein. We decided to use the Ssp DnaX, Ssp GyrB and Ter DnaE3 inteins as they were most positively characterised i.e. they showed the highest splicing activity.

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] 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).

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

[3] Brenzel, S. & Mootz, H. D. Design of an intein that can be inhibited with a small molecule ligand. J. Am. Chem. Soc. 127, 4176–7 (2005).

[4] iGEM team Queen's University, Kingston, ON, Canada 2014 https://2014.igem.org/Team:Queens_Canada/Project

[5] 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).

[6] Herrou, Julien, and Sean Crosson. Function, structure and mechanism of bacterial photosensory LOV proteins. Nature reviews microbiology 9.10 (2011). 713-723.

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

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

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

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

[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).

[12] Schwartz, E. C., Saez, L., Young, M. W. & Muir, T. W. Post-translational enzyme activation in an animal via optimized conditional protein splicing. Nat. Chem. Biol. 3, 50–4 (2007).