Motivation:

During the project search, we came across many interesting ideas and potential solutions. In the process of our research, the detection of tumor markers stood out in particular. Here, we especially addressed esophageal cancer and tried to find a solution for early detection of this type of cancer. In 2021 Watt et al. found specific marker proteins in the blood of esophageal cancer patients.[2] For these, we came up with several ideas on how to detect them. Another idea from a team member was a general detection system for the detection of low antigen amounts with a self-amplifying system. In the development of our project idea we wanted to use this general antigen-detection system to be able to measure the tumor markers for esophageal cancer. With this system, even the smallest amounts can be detected. This plays a particularly important role in various types of cancer, as early treatment is necessary to increase the chances of cure. Symptoms of esophageal cancer usually only appear at an advanced stage, so it is even more important to detect this cancer type as fast as possible to start the treatment early enough.

Background:

Esophageal cancer is the eighth-most common cancer globally.[3] Around 6,000 men and 2,000 women are diagnosed with this disease every year.[4] The problem with this type of cancer is often that it is only detected at an advanced stage and the chances of recovery are therefore reduced. About one in three esophageal cancers is detected at an early stage, so this type of cancer has a rather low chance of survival.

A differentiation is made between two different types of cancer. Once the more common squamous cell carcinoma and the adenocarcinoma. These esophageal cancers are mainly caused by typical risk factors, such as excessive alcohol consumption, smoking and overweight. The reason for this are mucosal changes in the esophagus.

Components:

Detection component:

Our cascade-like system is characterized by three components. The detection components which consist of scFv antibody fragments (scFv = single chain fragment variable). These can then be exchanged depending on the antigen to be detected. The only importance is that for this antigen scFv fragments are available that either bind to the antigen in a "sandwich-ELISA-like" manner (then two different scFv fragments are needed for compounds like toxins) or the antigen is presented multiple times on a particle (e.g. viruses). Therefore our system can detect eg.: coronaviruses, mycotoxins, snake venoms, biomarkers, etc..

[To learn more scFv fragments click here]

The information of antigen binding by the scFv fragments needs to be reported by the reporter component. For this internal signal transduction we choosed the TEV protease. Thats why our scFv fragments are linked to spli TEV protease halves. The full TEV protease is reconstituted ones the halves come into close proximity. This is facilitated by the antigen binding event.



[To learn more the TEV protease click here]

Reporter component:

Our Reporter component is made of fluorescence proteins (FP) so we can easily detect them. A FRET pair is linked together so the FRET state is on. The linker harbours a TEV protease recognition site. So it is cutted by TEV protease activity. If the linker is cutted the FPs can dissociate. If that happens the FRET state is off. This can be detected by the color shift since the former happening and the former quenched FP is now active. Alternatively the Reporter component can consist of reporter enzymes. But these have to be in such a way inactive, that the presence of TEV protease activity activates the enzyme. The following picture is given a example for our Reporter component.

Amplification component:

Now we come to the most important part of our system: the amplification component. It is based on a modified intein system. To be more specific: a split variant of an intein. This means that when the C- and N-part of this split intein are interacting with each other, the intein cuts itself out and the extein parts (in our case split TEV protease halves) are joined together to form an the active protease. To ensure that this interaction does not occur without prior activation, the sites of the intein halves that need to interact are masked. We make this possible by attaching a slightly modified part of the C-part of the intein to the N-part of the intein using a linker. The same principle applies to the C-terminal part of the intein and so, this created “cage” can prevent the interaction. But linker of these cages are harbouring TEV protease recognition sites. So ones a TEV protease cuts the linker, the cage can dissociate and the split inteins can then reconstitute to be active. At the end we get an amplification of the TEV protease activity by the amplification component and are thereby capable to detect small antigen amounts. In the following picture you can see a scheme of the mode of action of the amplification component.

Aim of our project:

Our final goal with LionDetect is, to make it easy for doctors to perform a quick and simple test. The test should be specific to the antigen and should detect even small amounts of the antigen. For this purpose we are building a toolbox, with which we try to build a small automated system for testing. Our box is integrated with an insert for filters and a smartphone which can then be used to record the FRET signal via camera. For this we have taken inspiration from the 2015 Bielefeld iGEM team with their box for the detection of knockout drops.

Literature research

ScFv Fragments:

A Fv (fragment variable) part is the smallest antibody part that is still capable of antigen binding consisting of one variable part from a heavy and one from a light antibody chain. Usually this both variable parts are not connected directly connected together. But in a scFv (single chain Fv) antibody they are connected with a linker. These conditions make the recombinant scFv antibody capable of binding antigens and being produced in microorganisms like E. coli[5][6][7]. ScFv antibodies can be easily created for many different antigens with the phage display technology[5][8]. Thats why we decided to use scFv antibodies for our antigen binding and recognition part of our detection system. We thought of a harmless antigen like gluten, that is easy to obtain (i.e. it is in flour) and a scFv antibody against peptic-tryptic digested gliadin in gluten was already developed[9]. Also other antigens could be hypothetically detectet by our system, since corresponding scFv fragments are consisting against e.g.: snake venoms[10][11], SARS-CoV-2[12][13].

TEV Protease:

Proteases are enzymes that catalyse the breakdown of proteins/peptides into smaller parts. Some of them have a high substrate specificity[14]. Since we want to use a protease activity for signal transduction in our system the protease has to be very specific. Therefore we found caspase 3, TEV protease (from the tobacco etch virus) and HRV 3C protease (from the human rhino virus) as suitable options[15][16][17] (Sakamoto et al., 2013; Phan et al., 2002; Ullah et al., 2016). To be able to switch the signal-transduction from an off-state to an on-state, a split protease is necessary. We did not find any caspase 3 split variants. But for the TEV protease and the HRV 3C protease split systems exist[18][19] (Wehr et al., 2006; Wang et al., 2021). It could be possible to create a split caspase 3 but since other options are available we did not want to take this challenge. The HRV 3C protease seems to be a more efficient option[17][19] (Ullah et al., 2016; Wang et al., 2021). Nevertheless we choosed the TEV protease, because it split variant was frequently used and an improved mutation exists[18][20][21][22]. Also the split TEV protease was used in iGEM multiple times (e.g.: the kill switch of "Physco Filter", TU-Munich iGEM team 2013, ; "Sonicell", Slovenia iGEM team 2016.)

FRET:

A FRET system is used to detect the released TEV protease. Förster (or Fluorescence) Resonance Energy Transfer (FRET) shows whether proteins are 1-10nm apart or further away. In this process, an excited donor fluorophore transfers energy to an acceptor fluorophore via a short-range (10 nm) dipole-dipole interaction.[23] Therefore, in the case of spatial proximity, the donor fluorophore is excited, the emission of the donor then excites the acceptor fluorophore and its emission can be seen. When the fluorophores are spatially separated, only the emission of the acceptor is visible, as the donor fluorophore is no longer excited.[24][25]

For detection in the test, the FRET pair of mTurqouise with emission spectra of Ex λ= 434 and excitation spectra of Em λ = 474 will be used as the donor, and EYFP with emission spectra of Ex λ= 513 and excitation spectra of Em λ = 525 as the acceptor. Both proteins are expressed on a plasmid and are linked to a TEv_protease interface. Upon excitation with a wavelength of 434 nm, resonance energy transfer takes place without TEV protease due to the spatial proximity, thus the emission of EYFP is visible. When the TEV protease is released, mTurquise and EYFP are separated, which interrupts the resonance energy transfer and the emission from the donor mTurquise becomes visible.

Reporter enzymes:

For our Reporter component we came along many different ideas. One of them was the use of reporter enzymes. Ones activated a reporter enzyme could make a huge color shift because one enzyme can process multiple educts to products. Our most promising idea was the use of astaxanthins (iGEM 2013, team Uppsala, https://2013.igem.org/Team:Uppsala/astaxanthin). This compound is found in salmon or shrimp and responsible for the red color. Its precursor is beta carotene a non red compound. It is produced in bacteria and the related genes can be expressed in E. coli. Our idea was to produce beta carotene in E. coli and use the next two needed enzymes in an on/off switchabel manner (e.g. with the help of inteins). Therefore an existing fusion protein could be used. It is connecting the two enzymes with a linker and by that making the astaxanthin production more efficient.[26] But we did not choose this system, because the enzymes are membrane bound. Since we want to use cell free extracts or purified proteins, we can not be sure that these enzymes still work in our system.

Additionally we thought of other Reporter enzymes like the famous luciferase or the RUBY system. But since the educts for the luciferase system are to expensive we did not choose that enzyme. RUBY is widely used ammong plants. We did not find any uses in Procaryota. Since it could be not possible to use the RUBY system without changes in procaryota and we did want to work with E. coli (a procaryot) we also did not choose the RUBY system.[27]

Inducible inteins:

Inteins are analogous to introns but at the protein level. They can cut their way out of a precursor protein and thus connect the flanked protein segments, the exteins. These then usually form a functional protein.

How do inteins work?

Inteins are proteins that possess autocatalytic activity. This enables them to remove themselves from the host protein by means of a process known as protein trans splicing (PTS) when they are embedded.[28] Normally, splicing is associated only with pre-mRNA splicing. This precursor protein contains three segments: a N-extein followed by the intein followed by a C-extein. During the protein splicing process, a significant change in the primary structure occurs, which removes the inteins and finally assembles the exteins.[28] The exteins are ligated back together by an amide bond and thereby form a mature polypeptide chain.[29] The mechanism of intein-mediated protein splicing reactions follows a sequence of multiple acyl transfer reactions required to cleave two peptide bonds. In this process, two exteins are joined and a new peptide bond is formed between the N-extein and the C-extein.[29]

What can inteins be used for?

Naturally split inteins have found widespread use in biotechnology due to their ability to drive the ligation of separately expressed polypeptides through protein trans-splicing (PTS). Therefore, scientist are working with the split variant of an intein. This means that the inactive C- and N-part of a split intein are interacting to reconstitute and thereby forming the active intein.

Variants of intein-splice mechanisms:

The protein splicing mechanism occours as a post-translational process. Thereby the intein catalyzes the excision from the flanking exteins, as well as the ligation of the exteins.[30] In our project, we applied protein trans-splicing (PTS) (as shown in the following CC-licensed figure ("D"), figure is a section of the original figure from Topilina and Mills, 2014.[30] ), which was induced by an active protease While significant progress has been made in the development of CPS systems based on the naturally split inteins, none of these offer modularity with respect to the type of triggers employed.

How to make inteins inducible?

To make protein trans-splicing inducible one should ensure that the interaction does not occur without prior activation. This can be made possible by a new construction of the split-intein halves. Just previously, it was discovered that electrostatic interactions between an unstructured cationic region in the C-terminal fragment (residues 1-13) and an unstructured anionic region in the N-terminal fragment (residues 51-102) initiate complementation of the split intein pair.[31]

Based on this discovery, the intein-constructs can be optimized by masking the regions important for interaction. For this purpose, the residues 51-102 of the N-terminal part and a linker were fused to the full-length C-terminal part of the intein and residues 1-13 of the C-terminal part as well as a linker were fused to full-length N-terminal part. This leads to a prevention of the interaction of both split-intein halves. To make the interaction inducible, a protease cleavage sequence is incorporated into the linker. Proteolytic removal of the cage sequences would trigger split intein association and hence PTS, as interaction of the split intein fusion partners is made possible again.[32]

How to use inteins as an amplification component?

In our iGEM project, caged proteins were used in the amplification component. Binding to an antigen by the detection component leads to the formation of an active TEV-protease. This active TEV-protase can now bind to the protease cutting site, allowing enzymatic cleavage and removal of the cages. The N- and C-part of the intein interact and thus the splicing reaction takes place. The intein cuts itself out and the extein parts are brought together - a new protein is formed. If a split-TEV-protease is used as the extein parts, a new, active protease is formed after the interaction, which can also cut at the linker of the caged-intein-halves. The amplification component thus drastically increases the signal, which is mediated by protease activity. The generated protases of the detection component as well as the amplification component can now pass on the amplified protease signal to the reporter component. In our project, the repoter component consists of a FRET pair which has a TEV-protease cutting sequence between the fluorophores. The proteolytic activity thus leads to a color change of the emitted light.

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