Genesis of Our Project, Cerberus

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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.

One head to bind to cellulose, the CBM3a

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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.

One head to bind biological macromolecules, streptavidin

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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).

We decided to use two versions of streptavidin: the native form which is tetrameric because we have data on its interaction with biotin as several iGEM project already described the system, and a monomeric version called mSA2² because we feared that the tetrameric streptavidin would cause aggregation of our protein Cerberus. We chose to work with this monomeric form because from all the reviewed monomeric protein, mSA has the highest biotin affinity (K_D = 2.8 nM³). 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.

In order to use our streptavidin head, biotinylated proteins are required. Thus, we designed a plasmid including the gene coding for the protein of interest in fusion with an additional 15-residues Avi-tag and a gene coding for BirA⁴. BirA is a small protein which is able to biotinylate proteins displaying an Avi-tag. Expression of biotinylated fluorophore (mTagBFP) and scygonadin (antibacterial peptide) were respectively performed in E. coli and P. pastoris.

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.
2. 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.
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.
4. 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.

One head to bind inorganic molecules, 4 - azido - L - phenylalanine

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In order to expand the versatility of our platform to non-biological protein or molecule, we introduced at the end of the C-terminus linker of the CBM3a, an unnatural amino acid. 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.

Incorporation of unnatural amino acid (UnAa) is well described¹. Briefly, an amber stop codon TAG is introduced at desired position in the DNA sequence coding for the platform Cerberus. Then, a second plasmid containing an engineered tRNA and aminoacyl-tRNA synthase from Methylococcus ianaschii dedicated to our NnAa and 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 NnAa will recognize the amber codon and translate it with the corresponding 4-azido-L-phenylalanine. In absence of NnAa, the amber stop codon is recognised as a classical stop codon and the translation is aborded.

Two different kind of click chemistry reactions could be used to bond an 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 catalyst². This second type of reaction allows to proceed quickly and to overcome the problem of the toxicity for the living cells.

We choose the second approach to bind fluorescein and paramagnetic beads to the 4-azido-L-phenylalanine head. Those compounds were bought directly with a dibenzocyclooctyne (DBCO) function attached to it. This way, the molecules were ready to be clicked to our Cerberus.

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. 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

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For further details about our system, please see our Design page.