Team:Freiburg bioware/Project

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<h4><a name="Fok_a"></a>In vitro assays</h4>
<h4><a name="Fok_a"></a>In vitro assays</h4>
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Revision as of 20:12, 21 October 2009

FREiGEM

   Project Report



  Summary

We focused our project on coupling and optimizing the characteristics of a restriction endonuclease with short oligonucleotides to develop a programmable and highly specific enzyme-oligo-complex. As a restriction endonuclease we chose the cleavage domain of the well studied endonuclease FokI from Flavobacterium okeanokoites. Normally FokI acts as a homodimer, each dimer divided in cleavage and restriction domain. Chandrasegaran and Miller have already made experiments to uncouple the cleavage and restriction domains of FokI and created a novel site-specific endonuclease by linking the cleavage domain to zinc finger proteins.
For our project we generated two Fok heterodimers (Miller, Nature biotech, 2007). For the catalytic active Fok partner, named Fok_a, the first 1158 nucleotides, i.e. the recognition domain, were deleted and glutamate 490 was switched to lysine (GAA->AAA) as well as isoleucine 538 to lysine (ATC->AAA) for the heterodimer formation. For the catalytic inactive Fok partner, named Fok_i, the heterodimeric amino acids glutamine 486 was switched to glutamate (CAA->GAA) and isoleucine 499 to leucine (ATC->CTG) and the catalytic amino acids aspartate 450 was switched to alanine (GAC->GCG) and aspartate 467 to alanine (GAT->GCG).

Association of linker FluA and Dig with DNA and Fok_a and Fok_i monomers
The two heterodimeric partners were fused to different anticalins binding different adapter molecules. Thus Fok_i is fused to anticalin on Fluorescein and Fok_a to anticalin on Digoxigenin. These adapter molecules are linked to oligonucleotides mediating the binding of the DNA site of interest. Now the heterodimerization comes into play. If the different Fok_i and Fok_a constructs bind their target oligos and come together, the inactive domain will serve simply as an activator of the active domain, cutting only one strand of the DNA. In our 3D models we showed that Fok domains are positioned in such a way that Fok_a will cut the DNA and Fok_i the modified oligonucleotide. Thus the inactivation of Fok_i allows the reuse of our oligonucleotides. Different linkers were designed and fused between cleavage domain and binding protein to test the optimal distance to preserve the most possible flexibility and most possible precision of the heterodimeric Foks.
complete universal restriction enzyme. Blue: DNA strand; Red: two 16bp long Oligos, tags as indicated in the picture. Ochre: Fluorescein A binding lipocalin; Orange: digoxigenin binding lipocalin; Yellow: tagged Base with C6 linkers and attached tags; light Blue: inactive FokI cleavage domain; Red: FokI active cleavage domain; Green: three catalytically active aminoacids; White: two Calcium ions
The place where all the cutting events will take place in our scenery is the thermocycler. Therefore all the ingredients, i.e. chromosomal or plasmid DNA of interest, modified oligonucleotides and the different heterodimers Fok_a and Fok_i are mixed together. At high temperatures the DNA will denaturate allowing the different partners to find each other, cut the DNA, and fall apart in course of one thermocycle. The whole procedure can be repeated undefinately. To reach this step, we need to improve the thermostability of our enzyme via phage display. Creating a universal restriction enzyme provides not only the possibility to improve routine cloning but also to enhance therapeutic gene repair via triplex technology. Many genetic diseases and especially ones arising from single nucleotide polymorphisms (SNPs) or monogenetic disease can be alleviated by the replacement of mutated genes using this method. To cut double stranded DNA the oligonucleotides have to be replaced by triple helix forming oligos (TFO). They can bind double-stranded DNA in homopurin- or homopyrimidine-rich areas. But developments are also made to widen the possible interaction domains of the DNA and hence make the TFOs as programmable as our conventional oligonucleotides. In case of the human genome of 3×10^9 bp size, a highly specific artifical endonuclease would be necessary to address the mutated gene explicitly. The used TFOs therefore have to possess a minimum length of 16 bp to cut just once in the human genome (4^16 bp = 4.3*10^9 bp).

Additionally, we provide an alternative way of binding by the coupling of the cleavage domain to the transcription factor Fos. This concept also includes the activity regulation by photo-switching. We
are also modifying an argonaute protein into a DNA endonuclease using phage display technics.



  Highlights


Modeling

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3D-Modeling

3D modeling was essential in our approach to construct a universal restriction enzyme.
Read more...

In vitro assays

After the cloning, expression and the purification of the Fok constructs we conducted several assays to analyse the activity of the enzyme. To establish the assay and as a reference for activity we used wildtype FokI. Binding of the modified nucleotides and enzymatic activity were tested with the Fok_i / Fok_a construct.
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Fok Monomer


In order to create a universal restriction enzyme we followed several ideas and models. The first and most promising idea we had was to create a Fok Monomer as it represents the optimal and easiest model for a universal restriction enzyme

If we are able to employ a monomeric enzyme, this protein would have a couple of advantages. Most importantly we would no longer need two separate oligonucleotides to achieve specific binding and cutting of the target DNA. Also only one protein has to be purified – thus saving time and money. A scientific advantage is the option to optimize the monomer by phage display. Using this technology, we would have the chance to create a thermostable, specific, universal restriction enzyme whose DNA binding activity is only created by a single oligonucleotide.

Our goal was firstly to create a Fok-monomer which is able to cut DNA without a primary dimerization step and secondly to show that our heterodimeric interface design works properly. To reach this, we had to clone all the required parts one after another, resulting in a huge fusion protein.
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Protein Expression and Purification


In order to produce and study our different protein constructs they had to be expressed in bacteria.  After cloning the individual parts into the pMA vector the complete expression products were then transferred into the pEx vector (see pEx vector ) and transformed into competent E. coli expression strains via heat shock. We used two different strains for the protein expression: E. coli BL21 de3 (Novagen) and E. coli RV308 (Maurer et al., JMB 1980). Both strains are suited for high-level protein expression.
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In vivo Expression



 Additionally to the in vitro assays with the directly purified programmable restriction endonuclease and isolated target  DNA, we developed an in vivo assay. This means the experiments with Fok constructs and DNA in living E. colis.
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AGO

The argonaute proteins represent one of our side projects in creating a universal programmable endonuclease.
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Alternative way of binding: Jun/Fos

We also thought of an alternative way of binding of the heterodimeric Fok to the DNA avoiding the necessity of labelled oligos and their binding proteins using the binding domain of a transcription factor as a new adapter. We focused on the binding domain of the activator protein-1 (AP-1) a crucial transcription factor implicated in numerous cancers. The protein is composed by a series of dimers. Nine homologues of the AP-1 leucine zipper region have been characterized, among them c-Fos, c-Jun and semirational library-designed winning peptides FosW and JunW. Via leucin zipper they interact among each other and with their basic region they bind DNA.
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Cloning strategy

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  Literature





contact:  freigem09@googlemail.com

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