P ROTEIN ENGINEERING WITH NON - NATURAL AMINO ACIDS

1.2.4. Termination

One of the three stop codons determines the termination of translation. Because there are not any tRNAs with complemental anticodons to stop codons, the ribosome stops at a stop codon. Then, release factors RF1 and RF2 place themselves at the A-site and RF3 removes RF1 or RF2 from the ribosome. EF-G together with the ribosome-recycling factor mediates the separation of the two ribosomal subunits and the release of mRNA and tRNA (Knippers 2006).

1.3. Protein engineering with non-natural amino acids

1.3.1. Selective pressure incorporation

Selective pressure incorporation (SPI) is based on the use of auxotrophic strains. This means that the cells are not able to biosynthesise one or more canonical amino acid themselves. Its roots lie in the classical experiment of (Cowie and Cohen 1957). They reported the incorporation of seleno-methionine into the whole proteome, using an E. coli seleno-methionine-auxotroph mutant strain. The bacterial growth rate was dependent on the external methionine supply. Therefore, it was possible to replace methionine by selenomethionine. In such cultures, the cells grew more slowly but exponential. Selenomethionine was found to completely and uniformly substitute methionine in all cellular proteins and thus, an “unnatural microorganism” was obtained.

With some exceptions as selenomethionine, all non-canonical amino acids that are not metabolic intermediates are toxic. However, it was observed that toxic analogues might serve as substrates in protein synthesis. If such toxic analogues are added together with their canonical counterparts in the growth media, usually lower incorporation levels in all cellular proteins are obtained. For

substitutions in single target proteins, this is a major problem to overcome in order to achieve full substitution. The use of auxotrophic strains provided a solution to circumvent toxic metabolic effects (figure 6). However, the auxotrophic approach for complete substitution of target proteins could be fully generalised to a single target protein only after the introduction of recombinant DNA

techniques. The basic requirements for a successful SPI-experiment include:

o Selection of a proper cell and expression system

o Control of fermentation conditions (for example the environment)

o Selective pressure for the replacement of the amino acid (for example the reassignment of a sense-codon in a single protein)

The amino acid analogues need to be sterically almost identical to the canonical ones and are called isosteres or surrogates. They have to fulfil three conditions:

o The uptake of the non-canonical amino acid o Its attachment onto the tRNA

o Its incorporation into the nascent polypeptide chain

In such approaches, the amino acid, which the cells cannot produce themselves, is supplied in restricted amounts for cellular growth. As the stationary phase is reached, the culture is transferred into another minimal medium depleted from the parental amino acid and with a high concentration

14 of the unnatural amino acid analogue. From this point on, the host cells serve only as a “factory” to produce the recombinant protein. In that way, the cells are forced to incorporate the unnatural amino acid instead of the missing canonical amino acid due to the lack of an absolute substrate specificity of the aminoacyl-tRNA synthetase. Thus, the toxicity can be circumvented in this straightforward way. An alternative would be to block biosynthetic pathways of the host cells by proper inhibitors (Budisa 2004).

Figure 6: Schematic overview of the selective pressure incorporation method; strong host auxotrophism and control of the fermentation conditions are crucial for effective unnatural amino acid incorporation. Figure is inspired by (Budisa and Biava 2014).

Azidohomoalanine (AHA) and homopropargylglycine (HPG) are two examples for methionine analogues, which can be introduced into proteins via SPI (figure 7). Bertozzi et al. have successfully demonstrated the incorporation of these two unnatural amino acids into the protein murine dihydrofolate reductase using methionine auxotrophic E. coli (Kiick, Saxon et al. 2002).

Figure 7: Structure of methionine (A) and its analogues azidohomoalanine (B) and homopropargylglycine (C)

1.3.2. (Amber) stop codon suppression

Another possibility to introduce unnatural amino acids into proteins is by stop codon suppression.

Therefore, a tRNA/aminoacyl-tRNA synthetase pair from another organism is needed, which recognises one of the three stop codons. This pair needs to be orthogonal to the host organism, which means that there are not any cross-reactions: The unnatural amino acid is not recognised by endogenous aminoacyl-tRNA synthetases, nor the orthogonal synthetase recognises one of the

15 canonical amino acids. Moreover, the stop codon is distinctly assigned to the unnatural amino acid.

One of the remaining two stop codons must then serve as stop signal.

Figure 8: Structure of pyrrolysine (A) and its derivatives (B-E); Plk (D), Pln (E)

In this study, the tRNACUA/pylRS pair from Methanosarcina barkeri was used, which recognises the amber stop codon UAG on the mRNA and assigns pyrrolysine to it. By introducing its genes into E. coli, it is possible to incorporate pyrrolysine into any recombinant protein opposite an amber stop codon (Blight, Larue et al. 2004). This is also possible for the structural similar derivatives of

pyrrolysine (figure 8). A schematic overview of the strategy to incorporate the pyrrolysine derivative Plk (figure 8D) into a target protein, is depicted in figure 9.

Figure 9: Schematic overview of Plk incorporation into a target protein as an example for amber suppression

An advantage of amber suppression over SPI is that the incorporation of non-canonical amino acids is exclusively opposite to the amber stop codon. Hence, canonical amino acids in the proteome are not replaced. The drawbacks of this method compared to SPI are the possible truncation of the target protein, due to a stop of the translational machinery at the amber stop codon and a tremendously decreased yield, if more than one amber stop codon is used within one target protein.

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