Genesis of Our Project, Cerberus

---

During our phase of brainstorming to pop up a project, we discussed around different subjects that would involve bio-functionalisation of a material. For example, we thought about a bandage made with cellulose fused with an antimicrobial peptide to accelerate skin wound healing. We also thought about producing dyes and grafting carbon nanotubes to cellulose for staining or adding conductivity properties. Each of these ideas would require a specific strategy. That is how we came up with the concept to generalise the functionalisation of cellulose and designed a generic and versatile platform protein named Cerberus.

Our platform protein is composed of three heads as the Ancient Greek mythological three-headed dog, keeper of the gate of the Underworld. The main head is a Carbohydrate Binding Module of the family 3 (CBM3a) from Clostridium thermocellum, well described to specifically bind to crystalline cellulose¹ (in green in the video above). Linked at the N-terminus of the CBM3, the second module (in red) is a streptavidin from Streptomyces avidinii displaying one of the strongest known linkage systems in Nature to biotinylated molecules². At the C-terminus of the CBM3a, we designed a linker incorporating an unnatural amino acid, 4-azido-L-phenylalanine (in blue in the video above), enabling the covalent bonds association of alkyne derivate molecule to Cerberus by click chemistry³. Therefore, our platform Cerberus will introduce versatility in cellulose functionalisation, with biological and/or with chemical components.

References

1. Morag E, Lapidot A, Govorko D, Lamed R, Wilchek M, Bayer EA, Shoham Y: Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum. Applied and Environmental Microbiology 1995, 61:1980-1986.
2. Nogueira ES, Schleier T, Durrenberger M, Ballmer-Hofer K, Ward TR, Jaussi R: High-level secretion of recombinant full-length streptavidin in Pichia pastoris and its application to enantioselective catalysis. Protein Expr Purif 2014, 93:54-62. DOI: 10.1016/j.pep.2013.10.015.
3. 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.

The Cellulose Binding Head

---

The first function of our platform is to bind to cellulose. The family 3 Carbohydrate Binding Module of type a (CBM3a) from Clostridium thermocellum¹ is part of a scaffold protein attached to the outer membrane of the bacteria and decorated with nine catalytic enzymes dedicated to the deconstruction of plant cell wall (Figure 1).

This enzymatic machinery known as the cellulosome², is one of the most effective degrading cellulose systems. Glucose sub-units of cellulose are recognised mainly by three aromatic amino acid side-chain displayed at the plan surface of the CBM3a. The CBM3a is composed of 159 amino acids. In our design, we also included the endogenous N- and C-terminus linkers (42 and 32 amino acids respectively) of the CBM3a in order to associate new domains to the CBM3a core part.

References

1. Morag E, Lapidot A, Govorko D, Lamed R, Wilchek M, Bayer EA, Shoham Y: Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum. Applied and Environmental Microbiology 1995, 61:1980-1986.
2. Lamed R, Setter E, Bayer EA: Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum. J Bacteriol 1983, 156:828-836.

The Streptavidin Head

---

At the N-terminus of the cellulose binding head, we fused a streptavidin module. Streptavidin is a 60 kDa homotetrameric protein composed of 512 amino acids, isolated from Streptomyces avidinii, and it is well known for its interaction with biotin and biotinylated proteins. With a dissociation constant of 10^-13 M, the interaction between streptavidin and biotin is the strongest non-covalent one known in Nature¹ (Figure1).

This streptavidin-biotin association is a very convenient way to link organic molecules to Cerberus. Indeed, most proteins and even other molecules such as DNA can be biotinylated. This should ensure wide possibilities to functionalise cellulose (enzymes or non-catalytic proteins such as some fluorophores).

References

1. Nogueira ES, Schleier T, Durrenberger M, Ballmer-Hofer K, Ward TR, Jaussi R: High-level secretion of recombinant full-length streptavidin in Pichia pastoris and its application to enantioselective catalysis. Protein Expr Purif 2014, 93:54-62. DOI: 10.1016/j.pep.2013.10.015.

The Click Chemistry Head

---

Some of the interesting new properties for cellulose are based on non-biological molecules which could not always be grafted on the streptavidin head. In order to expand the versatility of our platform to non-biological molecules, we introduced an unnatural amino acid at the end of the C-terminus linker of the CBM3a. The 4-azido-L-phenylalanine is a phenylalanine displaying an azide chemical function. This function reacts with an alkyne function, in a so called click chemistry reaction, in order to form a covalent bond (Figure 1).

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 chains. 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 and the mechanism is weel described¹. 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 used².

Briefly, for our system, an amber stop codon TAG is introduced at the desired position in the DNA sequence. Then, a second plasmid containing an engineered tRNA and aminoacyl-tRNA synthase from Methylococcus ianaschii dedicated to our UnAA and our amber codon is transformed into E. coli. During the expression of our protein, the medium is supplemented with 4-azido-L-phenylalanine. The tRNA charged with the UnAA will recognise the amber codon and translate it with the corresponding 4-azido-L-phenylalanine. In absence of UnAA, the amber stop codon is recognised as a classical stop codon and the translation is stopped.

Two different kind of click chemistry reactions could be used to bond the azide group to the alkyne group of the 4-azido-L-phenylalanine. The first one is the copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC). It takes place as its name suggests between the azide group of the unnatural amino acid and an alkyne group; the reaction is catalysed by Cu²⁺ ions. The main problem of this approach is the cytotoxicity of copper for cells, limiting its use in assays in vivo. The second type of click chemistry is the strain-promoted azide-alkyne cycloaddition reaction (SPAAC) which proceeds without the need of a catalyst³. This second type of reaction allows to proceed quickly and to overcome the problem of the toxicity for the living cells.

References

1. Young TS, Schultz PG: Beyond the canonical 20 amino acids: expanding the genetic lexicon. J Biol Chem 2010, 285:11039-11044. DOI: 10.1074/jbc.R109.091306.
2. Yang ST, Lim SI, Kiessling V, Kwon I, Tamm LK: Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein. Sci Rep 2016, 6:32866. DOI: 10.1038/srep32866.
3. 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.

Our system

---

To summarise, we aim to functionalise cellulose thanks to a generic and versatile platform to easily graft a wide range of organic and inorganic molecules to it. This Cerberus platform is structured to enable us to use two or three heads at the same time to further increase the possibility of cellulose functionalisation.

To know how we set up our system, check our Design page!