Oo

pdCpA

G NH2

pdCpA-uAA

Codon for residue of interest I m I r

Plasmid DNA containing the gene of interest

Codon for residue of interest I m I r

G NH2

pdCpA-uAA

Truncated tRNA

tRNA loaded with an unnatural amino acid

Xenopus oocyte mRNA

Truncated tRNA

tRNA loaded with an unnatural amino acid

Xenopus oocyte

Mutant protein evaluated with electrophysiology

Xenopus oocyte expressing mutant protein

Mutant protein evaluated with electrophysiology

Xenopus oocyte expressing mutant protein

FIGURE 4.8 Example of site-specific incorporation of unnatural amino acids into proteins expressed in Xenopus oocytes. © Chemical synthesis and aminoacylation of the dinucleotide, pdCpA; © Ligation of pdCpA-uAA to a truncated tRNA bearing the amber stop anticodon; © Mutation of the codon encoding the residue of interest into TAG amber stop codon by using site-directed mutagenesis; © Generation of mRNA through in vitro transcription; © Expression of mutant protein in Xenopus oocyte after coinjection of mRNA and tRNA, and the evaluation of the mutant protein with electrophysiology (two-electrode voltage-clamp recordings).

NHAlloc

NHAlloc

OH OH

Similarly, the technology has been applied to evaluate the importance of a proline residue for opening and closure of 5-HT3 receptors. It was hypothesized that cis-trans isomerism of the proline residue in the ion channel domain was important for the opening and closure of 5-HT3 receptors. This was investigated by the incorporation of proline analogs with either increased or decreased probability of cis-trans isomerism and there was a distinct correlation between this ability and the ability of the ion channel to open.

Although the technology has obvious and wide-ranging potential, it also has substantial limitations. Firstly, the generation of aa-tRNAs requires highly skilled persons in both chemistry and molecular biology. Secondly, the amount of protein generated is very low, thus exceptionally

Valine

Amide o

Valine

Amide

a-Hydroxy-valine (Vah)

Ester

a-Hydroxy-valine (Vah)

Ester

FIGURE 4.9 By using conventional genetic methods, changes in the protein backbone is not possible; however, by applying either unnatural mutagenesis or protein ligation strategies the amide backbone can be changed into, for example, an ester by using a-hydroxy acids rather than amino acids. This can be used to evaluate electronic effects of backbone carbonyl groups.

sensitive detection systems, such as electrophysiology or fluorescence, are required in these studies. Thirdly, the technology is generally limited to in vitro systems. Thus, in order to overcome some of these limitations, a modified method for incorporation of unnatural amino acids into proteins in vivo, was introduced. In this approach a custom-made pair of tRNA and aaRS is genetically introduced into a cell and the aaRS is engineered so that it only recognizes the unnatural amino acid and efficiently acylates the corresponding tRNA. Subsequently, the unnatural amino acid, which has to be nontoxic and cell permeable, is added to the growth media, taken up by the host organism and incorporated into the protein by the specific tRNA/aaRS pair. This technology, has been successfully applied in both yeast and eukaryotic cell, and allows the generation of proteins with an unnatural amino acid in reasonable yields. The primary challenge of this technology is that specific aaRS have to be generated for each unnatural amino acid, which is done by extensive mutational studies and rounds of positive and negative selections.

The technology has been applied to a number of model proteins, and has been used to specifically incorporate a glycosylated amino acid into myoglobin. In addition, a fully autonomous bacterium, E. coli, has been engineered so it could synthesize p-amino-phenylalanine, and a specific tRNA/aaRS pair was introduced, which allowed incorporation into myoglobin. The technology also holds commercial prospective, and a company (Ambrx) is developing protein therapeutics based on this technology.

A general limitation of these technologies is that the genetic code only contains three stop codons, which limits the theoretic numbers of different unnatural amino acids, that can be incorporated in a single protein to two. To overcome this limitation, Sisido and colleagues have explored an alternative strategy using extended codons and frameshift suppression. In this approach, an mRNA containing an extended codon consisting of four or five bases is being read by a modified aa-tRNA containing the corresponding extended anticodon. In certain species, some naturally occurring codons are rarely used and the amount of their corresponding tRNA is low. This has been used in the design of four-base codons, which are derived from these rarely used codons, to minimize the competition between the four-base anticodon tRNA and endogenous tRNA. The four-base codon technique has been used to incorporate unnatural amino acids into proteins in E. coli. It has also been used to incorporate two different unnatural amino acids into two different sites of a single protein showing that four-base codons are not only orthogonal to their host organism but also to each other.

Was this article helpful?

0 0

Post a comment