DESIGN

Our System

To exploit the potential of cellulose as a material capable of being functionalised, we designed a three headed linker protein named Cerberus. The protein will allow us to bind an infinity range of molecules to cellulose with a broad range of properties, thanks to its three fixating protein structures representing the three heads of the system.

We ran our tests on two different types of cellulose: a commercial Avicel was used to make colloidal particles named Regenerated Amorphous Cellulose (RAC), and bacterial cellulose produced by the bacteria Gluconacetobacter hansenii. Our three proteins (Sirius, Orthos and Cerberus) were designed to be produced in two chassis: Escherichia coli and Pichia pastoris. We chose to work with both a bacterium and a yeast because they both have their advantages. E. coli is a well-studied microorganism with a fast growth rate while P. pastoris is antibiotic-resistant, an ability that comes in handy when wanting to produce and antibacterial peptide, and it has a strong secretion capability. In order to validate our protein design we built a workflow, integrating in silco , in vivo and in situ validation steps as showed on figure 1.

A generic square placeholder image with rounded corners in a figure. — **Figure 1:** Overview of ou experimental procedure

Choosing the First Head

The platform binds to every type of cellulose through its protein domain of the type 3 Carbohydrate Binding Module (CBM3a) family. The Imperial iGEM team from 2014 proved that of all the CBMs, it has one of the highest specificities for crystalline cellulose. Also, as it comes from the bacterium Clostridium thermocellum, it is thermostable.

Choosing the Second Head

The first linker consists in a streptavidin head, which enables us to bind biotin and biotinylated compounds by using the strong affinity these two molecules have for each other. This interaction is in fact one of the strongest non-covalent bonds found in Nature with a dissociation constant of 10^-13 M, which is 10³-10⁶ greater than any interaction between a ligand and its specific antibodies.

Of all the interacting pairs known and used today, we chose the biotin-streptavidin because of its appealing characteristics that can be used in a whole range of applications¹. The high affinity ensures that once formed the complex will not be changed by pH variations or washes, and that the binding is highly specific and will only specifically target certain molecules. When attached to macromolecules, thanks to its small size, biotin does not alter their biological activity or other properties. Finally, what makes streptavidin a great choice for our fusion protein is that it is very stable and its ability to bind to biotin is not easily destroyed as it can survive hard conditions.

Also, the fact that biotinylated compounds such as fluorophores, enzymes and proteins are commercially available encouraged us to use this system in our project. All these aspects also explain why the use of the streptavidin-biotin interaction was developed in the field of biotechnology. It opens a broad range of applications, especially on immunological and nucleic acid hybridization assays.

During our experiments, we decided to use two types of streptavidin: the wild-type which is tetrameric because we have data on its interaction with biotin as several iGEM teams already described the system, and the mSA2 which is monomeric and the result of a fusion of streptavidin and rhizavidin because we feared that the tetrameric streptavidin would cause aggregation of Cerberus². Both have their pros and cons, that is why we chose to work with both. Due to its capacity to bind four molecules of biotin per tetramer, the wild-type has an unmatched affinity for biotin. However, the monomeric version mSA2 has a shorter nucleic sequence and it is less prone to have non-specific interactions. Of all the existing non-tetrameric streptavidin, mSA has the highest biotin affinity (K_D = 2.8 nM)³, which increases its ability to detect biotinylated compounds. Additionally, mSA2 is easier to produce as our experiments have shown us, which explains why we did not conduct all our proofs of concept with the tetrameric type.

References

Diamandis EP, Christopoulos TK: The biotin-(strept)avidin system: principles and applications in biotechnology. Clin Chem 1991, 37:625-636.
Lim KH, Huang H, Pralle A, Park S: Stable, high-affinity streptavidin monomer for protein labeling and monovalent biotin detection. Biotechnol Bioeng 2013, 110:57-67. DOI: 10.1002/bit.24605.
Qingqing Li QL, Xiaojian Ma, Yaping Wang, Mengjie Dong, Zhen Zhang and Lixin Ma: High-level expression of biotin ligase BirA from Escherichia coli K12 in Pichia pastoris KM71.
Pickens CJ, Johnson SN, Pressnall MM, Leon MA, Berkland CJ: Practical Considerations, Challenges, and Limitations of Bioconjugation via Azide-Alkyne Cycloaddition. Bioconjug Chem 2018, 29:686-701. DOI: 10.1021/acs.bioconjchem.7b00633.

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.^1s/sup>

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

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 Avidin Protein Family: The Strongest Non-Covalent Bond

Domain Diversity

During our experiments, we used two types of streptavidin: the wild-type, which is tetrameric, and the mSA2, which is monomeric and the result of a fusion of streptavidin and rhizavidin. Both have their pros and cons, that is why we chose to work with both. Due to its capacity to bind four molecules of biotin per tetramer, the wild-type has an unmatched affinity for biotin⁶. However, the monomeric version mSA2 has a shorter nucleic sequence and it is less prone to have non-specific interactions. Of all the existing non-tetrameric streptavidin, mSA has the highest biotin affinity (K_D = 2.8 nM)⁶, which increases its ability to detect biotinylated compounds. Additionally, mSA2 is easier to produce as our experiments have shown us, which explains why we did not conduct all our proofs of concept with the tetrameric type.

Biotinylation Diversity and Usages

Of all the interacting pairs known and used today, we chose the biotin-streptavidin because of its appealing characteristics that can be used in a whole range of applications⁷. First, this interaction is one of the strongest non-covalent bond found in Nature with a dissociation constant of 10^-13 M, which is 10³-10⁶ greater than any interaction between a ligand and its specific antibodies. The high affinity ensures that once formed the complex will not be changed by pH variations or washes. Secondly, this strong affinity ensures that the binding is highly specific and will only specifically target certain molecules. When attached to macromolecules, thanks to its small size, biotin does not alter their biological activity or other properties. Finally, what makes streptavidin a great choice for our fusion protein is that it is very stable and its ability to bind to biotin is not easily destroyed as it can survive hard conditions.

Also, the fact that biotinylated compounds such as fluorophores, enzymes and proteins are commercially available encouraged us to use this system in our project. All these aspects also explain why the use of the streptavidin-biotin interaction was developed in the field of biotechnology. It opens a broad range of applications, especially on immunological and nucleic acid hybridization assays.

Experimental Plan: Orthos

Orthos is our second protein used to validate our project. It is the result of the fusion of binding molecular platform, 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. After validating it by sequencing, we produced it in E. coli BL21 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. To do that, we fused mTagBFP with an AviTag, and cloned it in pETDuet1. The AviTag will be recognised by a small protein called BirA and the molecule will be biotinylated by it. Once the system was transformed into E. coli BL21 and induced with IPTG, we used the cellular lysate to prove the affinity of Orthos for biotinylated compounds by measuring the fluorescence.

UnNatural Amino Acid

Expanding the Genetic Code with the Orthogonal Machinery of Incorporation

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.

Cerberus: The Versatility of a Simple System

Primary Usage: The Tricephalic Protein

Cerberus was first thought for binding different types of compounds at the same time thanks to its two very unlike linkers. The streptavidin linker can bind biotin and biotinylated compounds while the UnAA-containing linker will click molecules that bear an alkyne function. The main advantage of such a system is that we can bring not one but two varied and distinct functions. This way, the possibilities of functionalizing cellulose and bringing new innovative materials are expanded.

Secondary Usage, or How to Recycle?

Imagine that someone wants to double the amount of a molecule bound onto a surface. We have thought about using a biological “adapter”, DBCO-Biotin. This compound can be purchased, and it allows us to turn one linker into another or to form dimers and more of Cerberus. Components with alkyl functions can now be grafted on the streptavidin linker while the streptavidin head of the second Cerberus can bond on the AzF linker.