Expanding the genetic code for biological studies

Abstract

Using an orthogonal tRNA-synthetase pair, unnatural amino acids can be genetically encoded with high efficiency and fidelity, and over 40 unnatural amino acids have been site-specifically incorporated into proteins in Escherichia coli, yeast, or mammalian cells. Novel chemical or physical properties embodied in these amino acids enable new means for tailored manipulation of proteins. This review summarizes the methodology and recent progress in expanding this technology to eukaryotic cells. Applications of genetically encoded unnatural amino acids are highlighted with reports on labeling and modifying proteins, probing protein structure and function, identifying and regulating protein activity, and generating proteins with new properties. Genetic incorporation of unnatural amino acids provides a powerful method for investigating a wide variety of biological processes both in vitro and in vivo.

Figure 1

A general method for genetically encoding unnatural amino acids in live cells.

Figure 2

Double-sieve selection with a mutant library for evolving orthogonal tRNAs or unnatural amino acid-specific synthetases. (A) Evolving orthogonal tRNAs in E. coli. The negative selection uses toxic barnase gene containing two TAG stop codons at permissive sites. tRNAs non-orthogonal to E. coli synthetases will be charged with amino acids and suppress the TAG codons to produce a functional barnase, which kills the cell. The positive selection uses the β–lactamase gene containing a TAG codon at a permissive site. tRNAs charged by the cognate synthetase and functional in translation will suppress the TAG codon and express active β–lactamase, which enables cells to survive in ampicillin. Cells containing non-functional tRNAs will be killed by ampicillin. (B) Evolving orthogonal synthetases to be specific for an unnatural amino acid in E. coli. The positive selection uses the chloramphenicol acetyl transferase (CAT) gene containing a permissive TAG stop codon in the presence of the unnatural amino acid. Mutant synthetases capable of charging the orthogonal ${\text{mutRNA}}_{\text{CUA}}^{\text{Tyr}}$ with the unnatural or any natural amino acid will express full length CAT, enabling cells to survive in chloramphenicol (Cm). The negative selection uses the barnase gene containing permissive TAG codons. Synthetase mutants active toward natural amino acids will charge the ${\text{mutRNA}}_{\text{CUA}}^{\text{Tyr}}$ to suppress TAG codons in the barnase gene, leading to cell death. In the absence of the unnatural amino acid, synthetase mutants specific for the unnatural amino acid will survive the negative selection. To improve activity and specificity, initial synthetase gene hits can be recombined using DNA shuffling and subject to next round of selection.

Figure 3

General methods for efficient expression of prokaryotic tRNAs in eukaryotic cells using special Pol III promoters. (A) Gene elements for tRNA transcription in E. coli and eukaryotic cells. (B) Expression of prokaryotic tRNAs in yeast using an external SNR52 or RPR1 Pol III promoter, which contains the consensus A- and B-box sequences and is post-transcriptionally cleaved from the primary transcript. (C) A type-3 Pol III promoter, such as the H1 promoter, with a defined transcription initiation site is used to express prokaryotic tRNAs in mammalian cells.

Figure 4

Structures for unnatural amino acids discussed in text.

Figure 5

Protein labeling and modification through the genetically incorporated chemical handles. (A) A general method for preparing glycoprotein mimetics with defined glycan structure using the keto group. (B) Protein modification with PEG through a copper-catalyzed cycloaddition reaction (top) and with fluorescein through Staudinger ligation (bottom). (C) Protein modification through phenylselenocysteine.

Figure 6

Unnatural amino acids to probe the inactivation mechanism of ion channel Kv1.4. o-Methyltyrosine or dansylalanine extends the side chain length of tyrosine, which impedes the inactivation peptide from threading through the side portal of the ion channel and abolishes the fast inactivation, as shown in the current-time curve.

Figure 7

Regulation of protein activity using photo-responsive amino acids. (A) Photocaged serine is used to control when the target serine is regenerated and phosphorylated. (B) The photo-isomerizable p-azophenyl-phenylalanine is used to control the binding of CAP to its promoter determined by gel mobility shift assay.