The protein world is vast and seems endless. Even natural proteins, composed of the 20 proteinogenic α-amino acids found in all organisms, have seemingly infinite possibilities and are not yet fully understood. Nevertheless, it is intriguing to search for possibilities of endowing these proteins with new, unnatural functions.
With a few exceptions, the life we know is composed of 20 L-amino acids. These amino acids can be modified, for example, by phosphorylation or oxidation. Non-proteinogenic amino acids are called non-canonical amino acids (ncAAs). The ncAAs can endow living cells with new structural, chemical and physical properties [51]. One strategy to expand the genetic code of Escherichia coli (E. coli) is to use auxotrophic strains and replace the amino acid with close structural homologs [51]. Another way to introduce ncAAs into a living organism requires some prerequisites. An aminoacyl-tRNA synthetase (aaRS) and a tRNA (orthogonal) derived from other species must be introduced into the organism. The tRNA must not be recognized by the aaRSs of the organism, the same applies to the introduced aaRS and tRNAs of the cell. In addition, the new tRNA must introduce the ncAA via a codon that does not encode any of the 20 natural amino acids, and the ncAA must be taken up by the cell [51][52][53]. The requirements described above are fulfilled by E. coli in combination with the evolved tyrosyl-tRNA/synthetase pair of Methanococcus jannaschii. Amber stop codon (UAG) suppression technology is used for this purpose, whereby the codon is recognised by the tRNA, and the loaded ncAA is incorporated (Figure 5). There are also other pairs from other organisms available [51][54][55]. Through the incorporation of ncAAs, it is possible to give proteins new properties: photocrosslinking and chemical crosslinking, increasing of the melting temperature, formation of longer disulphide bridges, click chemistry and protein immobilisation, as well as many others [56][57][58].
Figure 1: Schematic representation of an aminoacyl-tRNA synthetase (aaRS), a tRNA (orthogonal) and an ncAA. An aminoacyl-tRNA synthetase loads an orthogonal tRNA with a specific non-canonical amino acid (ncAA). The tRNA loaded with the ncAA recognises the amber stop codon with its anti-codon and the loaded ncAA can be incorporated into the protein. Figure was adapted from [14].
For the project we used two different ncAAs and aminoacyl-tRNA synthetases with their corresponding tRNAs under different conditions to get a broad understanding of the efficiency of amber stop codon suppression. The first amino acid is 4-azido-L-phenylalanine (p-Azf), which can be used for photolabeling via click chemistry. For this purpose, the amino acid is incorporated into a protein and an alkyne fluorescent dye is added. The azido group of the amino acid reacts with a fluorescent dye via click reaction, resulting in fluorescently labeled proteins, which can be detected [14][57]. Detecting and understanding interactions between different proteins is one of the most common but also most difficult questions in biology, especially when these interactions are weak or pH dependent [57]. The second amino acid used, 4-benzoyl-l-phenylalanine (p-Bpf) acts as a photocrosslinker and makes crosslinking possible, even in living cells [59]. To crosslink proteins, the amino acid must be irradiated by UV light with a wavelength of 365 nm [57].
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iGEM Freiburg 2022 presents
chAMBER
