Validating Our System

---

Validating each head of our system was the first step we had to go through, even before thinking about adding a whole range of properties. In order to do so, we designed three proteins: Sirius, Orthos and Cerberus. The aim of these proteins is to validate our system one head after the other.

Experimental Plan: Sirius

---

The first step of our experimental plan was to prove the affinity of our CBM3a to cellulose. To do that, we designed our first protein, Sirius. It was constructed by fusing CBM3a to mRFP1, which is an engineered version of DsRED but with a monomeric characteristic. This part was cloned into pET28 containing a pT7Lac promoter and a C-terminus His-tag. Once the cloning was validated by sequencing, we chose to transform it into E. coli BL21 DE3 because of its high protein production yield. Once produced, Sirius was purified by Immobilized Metal Affinity Chromatography (IMAC) on cobalt resin column, and the different fractions we collected were analysed on SDS-PAGE to make sure our protein was produced at the expected size.

Then, the binding affinity to cellulose was proved by using regenerated Avicel in a method called pull-down assay. The pull-down assay consists in separating soluble and insoluble compounds by using centrifugation. Two controls were used during the experiment: one with the Tris-HCl buffer and the Avicel, and the other with mRFP1 alone and the Avicel. These controls allow us to prove that the fluorescent protein binds to cellulose through our CBM3a and not directly. We then measured the fluorescence intensity in our tubes after several washes.

Experimental Plan: Orthos

---

After validating the affinity of our CBM3a to cellulose, we moved on the validation of the activity of the streptavidin head with a second protein called Orthos (Figure 2). Orthos is the result of the fusion of binding the molecular domain, CBM3a, and the streptavidin linker, mSA2, and this part ends with an amber stop codon, preventing the His-tag from the pET28 environment to be translated.

Between the C-terminus of the streptavidin amino acid sequence and the N-terminus of the linker of the CBM3a, we introduced a TEV-protease site. This insertion may provide some strategy of releasing of the streptavidin and the biotinylated protein interacting with it if required, simply by incubating our complex with TEV protease.

After validating Orthos by sequencing, we produced it in E. coli BL21 DE3 and purified it by using the cellulose affinity proven with Sirius.

To study its affinity for biotinylated proteins, we needed to in vivo biotinylate proteins. Thus, we fused mTagBFP with an Avi-tag, and cloned it in pETDuet1 which already had the sequence coding for BirA. BirA is a small protein able to recognise the 15-residues Avi-tag and to biotinylate the molecule attached to it. Once the system was transformed into E. coli BL21 DE3 and induced with IPTG, we used the cellular lysate to prove the affinity of Orthos for biotinylated compounds by measuring the fluorescence after a pull-down assay. The controls used for Orthos were the same than for Sirius: one with the Tris-HCl buffer, and one with the biotinylated fluorescent protein.

Experimental Plan: Cerberus

---

Now that two heads were validated, we still had to prove the activity of unnatural amino acid. For that, we used a third protein called Cerberus (Figure 3). Cerberus is a protein of 380 to 386 amino acids (depending on the type of streptavidin chosen) and 41 kDa. The affinity of the CBM3a to crystalline cellulose is around 2.9x10⁷ M^-1.¹

The construction is similar to Orthos’ but to incorporate the UnAA, we had to hack into the genetic code. To do so, we brought to E. coli an orthogonal aminoacyl tRNA synthase (aaRS)/tRNA pair. The amber stop codon present in our construction is now recognised by the tRNA and it becomes an incorporation site for the 4-azido-L-phenylalanine (AzF).

This strategy was thought for specific purification because the His-tag will only be attached to AzF-containing proteins. Once validated, the part was co-transformed into E. coli BL21 DE3 with pEVOL-AzF, a plasmid containing tRNA and aaRS from Methylococcus ianaschii. We purified the production by using IMAC purification on cobalt column.

To validate the activity of the UnAA, we used fluorescein grafted with DBCO by using SPAAC. This compound clicked with Cerberus’s head and after several washes we measured the difference of fluorescence between cellulose samples containing or not our Cerberus protein with the fluorophore.

References

---

1. Tomme P, Boraston A, McLean B, Kormos J, Creagh AL, Sturch K, Gilkes NR, Haynes CA, Warren RA, Kilburn DG: Characterization and affinity applications of cellulose-binding domains. J Chromatogr B Biomed Sci Appl 1998, 715:283-296.

The Applications of Our System

---

Paramagnetic Beads

---

We first aimed to bind paramagnetic beads on the UnAA with SPAAC reaction, imagining that we could this way create magnetic cellulose that could be used as a new way to extract compounds in cells, for example, by biotinylating and binding the molecule of interest on the streptavidin head. We bought beads already attached to a DBCO function, and we put them in presence of our Cerberus under agitation overnight to allow the click reaction to occur. The next day, a pull down-assay is made, and the magnetism is observed after several washes by putting the tubes next to a magnet and by observing the functionalised cellulose moving towards it. The three controls were the Tris-HCl buffer, Cerberus alone, and the paramagnetic beads alone.

Antibacterial Peptide, the scygonadin

---

The thought of having an antibacterial bandage is very appealing and it becomes more interesting with the TEV site we added. This small site enables us to release the antibacterial molecule directly into the wound just by adding a protease. After a week of culturing Pichia pastoris engineered to produce the scygonadin, the supernatant is collected after centrifugation and put with our Cerberus overnight under agitation. A pull-down assay is realised to purify the complex Cerberus-scygonadin. Then, the sample is put on a paper disk on a Petri dish coated with bacteria. The inhibition halos are observed the next day after incubation (Figure 2). The three controls are Cerberus, scygonadin and an antibiotic.

Testing the TEV site

---

This experiment is the same as the validation of our Orthos protein, except that one of the washes of the pull-down assay is replaced with a solution containing the TEV protease (see our Experiments page for further details) and that we use our Cerberus protein instead. The protein is incubated with the protease for 2 hours at 30°C. The fluorescence is then observed after centrifugation, showing that it is now present in the supernatant and not in the pellet as it used to be, showing and the biotinylated fluorescent protein was cut off from the rest of our protein. In the elution fraction, no fluorescence should be observed and measured as it was in the supernatant, and hence replaced with Tris-HCl buffer. The negative control is the biotinylated protein with cellulose.

Binding Two Different Molecules at Once

---

Finally, we aimed to bind different molecules on each head at the same time. For that, we decided to bind the paramagnetic beads on the UnAA, as it normally already have been proven to work, and a fluorescent protein bound to an azide function called FAM-azide on the streptavidin head. To bind the FAM-azide on the streptavidin head, the molecule DBCO-Biotin was used. The click chemistry between the UnAA and the paramagnetic beads was made overnight, then a pull-down assay is realised to purify it. The molecules DBCO-biotin and FAM-azide are then added, and a pull-down assay is made. The magnetism is once again observed with a magnet, but this time on an UV bench so we can observe the fluorescent cellulose.

Graphene and Carbon Nanotubes

---

Binding graphene and carbon nanotubes to cellulose could bring interesting physical properties to it like rigidity. To bind these two compounds to our UnAA, they first needed to be activated by a reaction of diazotisation. The activation is validated by a Thermogravimetric Analysis (TGA) where the mass loss is measured to prove that the function was added in the first place. The reaction between the alkyne group and the azide function of the UnAA is still a click-chemistry but it is now copper-catalyzed (CuAAC). We only had time for the activation of the graphene and carbon nanotubes, hence we did not conduct the CuAAC reaction to functionalise our cellulose.

FRET

---

To validate the distance between the streptavidin and the AzF heads calculated by 3D molecular modelling, we thought about using Förster resonance energy transfer (FRET). We aimed to bind fluorescein on the AzF head and biotinylated BFP on the streptavidin head. Fluorescein would have been the donor chromophore and BFP the acceptor chromophore. The distance between the two heads is around 6nm (see our Modelling page), so the FRET would have been observed as its range is usually between 1 and 10nm. Due to a lack of time, we have not been able to perform this test.

Cerberus: the Versatility of a Simple System

---

Primary Usage: the Tricephalic Protein

---

In many cases, proteins are built within organisms by combining the 20 common amino acids. To genetically encode more amino acids, referred to as unnatural, opens the possibilities of controlling the chemical functions of proteins and thus expanding their chemical space. For example, proteins can be expressed with novel side chains⁸. The strategy is to use the cellular machinery to introduce these unnatural amino acids (UnAAs). Today, more than 50 UnAAs have already been incorporated in different microorganisms. This is done by using orthogonal amino acyl tRNA synthetase (aaRS)/tRNA pairs, and an unassigned or reassigned codon. The 20 common amino acids are encoded by 61 triplet codons, which leaves the three stop codons (TAG, amber; TAA, ochre; and TGA, opal) to serve to specify the UnAA. For the 4-azido-L-phenylalanine, the amber stop codon was used⁹.

4-Azido-L-Phenylalanine: The Click Chemistry

---

At the beginning of the 21^st century, the emergence of the reaction called “click chemistry” revolutionized bioconjugate chemistry. Thanks to this, coupling molecular fragments to synthesize conjugates has become easier. Following that, biorthogonal reactions were developed. The most widely used is the copper-catalyzed azide-alkyne cycloaddition reaction, shortened as CuAAC¹⁰. Catalysed by Cu(I) ions, this reaction involves the azide group of our UnAA and an alkyne group. The main problem of this method is the cytotoxicity of copper on cells limits its use in assays that take place in the cellular environment. To circumvent this problem, the strain-promoted azide−alkyne cycloaddition reaction, shortened as SPAAC, was introduced in 2004 and have been more and more used nowadays. This reaction presents the advantage of not needing a catalyst, so it not toxic for the cells, and it can be implemented in physiologic pH condition (around 7.5).

Experimental Plan: Cerberus

---

Finally, the third protein we produced was Cerberus. The construction is similar to Orthos’ but to incorporate the UnAA, we had to hack into the genetic code. To do so, we brought to E. coli an orthogonal aaRS/tRNA pair. The amber stop codon present in our construction is now recognised by the tRNA and it becomes an incorporation site for the 4-azido-L-phenylalanine (AzF). This strategy was thought for specific purification because the His-tag will only be attached to AzF-containing proteins. Once validated, the part was co-transformed into E. coli BL21 with pEVOL-AzF, a plasmid containing tRNA and aaRS from Methylococcus ianaschii. We purified the production by using IMAC purification on cobalt column.

To validate the activity of the UnAA, we used fluorescein grafted with DBCO by using SPAAC. This compound clicked with Cerberus’s head and after several washes we measured the difference of fluorescence between cellulose samples containing or not our Cerberus protein with the fluorophore.

Functionalising Bacterial Cellulose in a Reactor

---

After validating each head separately and the ability of our platform to functionalise commercial cellulose, we had to make sure that the bacterial cellulose produced by Gluconacetobacter hansenii could be functionalised. To do so, we agitated a thin piece of bacterial drown in a purified fraction of Sirius and washed it several times to observe that the cellulose remains pink, demonstrating that our protein remains bound to it. The negative control used is the bacterial cellulose drown in the same fluorescent protein but without fusing it with our CBM3a. Normally, after washing this cellulose, the colour pink should not remain as the protein does not naturally bind to cellulose.

Once that the test is validated, we can imagine putting our chassis Pichia pastoris and Gluconacetobacter hansenii together in a reactor, so they can respectively produce our linker with the desired function and the bacterial cellulose. Tests were run to see if the two microorganisms could grow together but due to the acidification of the medium by G. hansenii, the results were not conclusive. This part remains to be optimised and other experimental conditions needs to be tested. For example, as P. pastoris growth rate is slower than G. hansenii, we could test putting one after the other, and we could have a system permitting to keep the pH stable.