Crucial Optimization of Translational Components towards Efficient Incorporation of Unnatural Amino Acids into Proteins in Mammalian Cells

The ability to site-specifically incorporate unnatural amino acids (UAAs) into proteins is a powerful tool in protein engineering. While dozens of UAAs have been successfully introduced into proteins expressed by Escherichia coli cells, it has been much more challenging to create tRNA and tRNA-Synthetase pairs that enable UAAs incorporation, for use in mammalian systems. By altering the orthogonality properties of existing unnatural pairs, previously evolved pairs for use in E. coli could be used in mammalian cells. This would bypass the cumbersome step of having to evolve mutant synthetases and would allow for the rapid development of new mammalian pairs. A major limitation to the amount of UAA-containing proteins that can be expressed in the cell is the availability of UAA-charged orthogonal suppressor tRNA. By using a natural mammalian tRNA promoter, the amount of functional suppressor tRNA can be greatly increased. Furthermore, increasing recognition of the suppressor tRNA by the mutant synthetase will ultimately lead to the appearance of more UAA-charged tRNA.     

Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: minimizing misacylation

The incorporation of unnatural amino acids site-specifically is a valuable technique for structure-function studies, incorporation of biophysical probes, and determining protein-protein interactions. THG73 is an amber suppressor tRNA used extensively for the incorporation of >100 different residues in over 20 proteins, but under certain conditions THG73 is aminoacylated in vivo by endogenous aminoacyl-tRNA synthetase. Similar aminoacylation is seen with the Escherichia coli Asn amber suppressor tRNA, which has also been used to incorporate UAAs in many studies. We now find that the natural amino acid placed on THG73 is Gln. Since the E. coli GlnRS recognizes positions in the acceptor stem, we made several acceptor stem mutations in the second to fourth positions on THG73. All mutations reduce aminoacylation in vivo and allow for the selection of highly orthogonal tRNAs. To show the generality of these mutations, we created opal suppressor tRNAs that show less aminoacylation in Xenopus oocytes relative to THG73. We have created a library of Tetrahymena thermophila Gln amber suppressor tRNAs that will be useful for determining optimal suppressor tRNAs for use in other eukaryotic cells.     

Misacylation of yeast amber suppressor tRNA(Tyr) by E. coli lysyl-tRNA synthetase and its effective repression by genetic engineering of the tRNA sequence

Through an exhaustive search for Escherichia coli aminoacyl-tRNA synthetase(s) responsible for the misacylation of yeast suppressor tRNA(Tyr), E. coli lysyl-tRNA synthetase was found to have a weak activity to aminoacylate yeast amber suppressor tRNA(Tyr) (CUA) with L-lysine. Since our protein-synthesizing system for site-specific incorporation of unnatural amino acids into proteins is based on the use of yeast suppressor tRNA(Tyr)/tyrosyl-tRNA synthetase (TyrRS) pair as the "carrier" of unusual amino acid in E. coli translation system, this misacylation must be repressed as low as possible. We have succeeded in effectively repressing the misacylation by changing several nucleotides in this tRNA by genetic engineering. This "optimized" tRNA together with our mutant TyrRS should serve as an efficient and faithful tool for site-specific incorporation of unnatural amino acids into proteins in a protein-synthesizing system in vitro or in vivo.     

The phenylalanine tRNA from Mycoplasma sp. (Kid): a tRNA lacking hypermodified nucleosides functional in protein synthesis

Phenylalanine tRNA from Mycoplasma sp. (Kid) was purified and characterized. The tRNA can be aminoacylated by phenylalanyl-tRNA synthetase from both Mycoplasma and E. coli. In a tRNA-dependent cell-free E. coli amino acid incorporating system programmed with poly U pure Mycoplasma tRNA(Phe) was fully active in promoting phenylalanine incorporation, even in direct competition with homologous E. coli tRNA(Phe). Since the Mycoplasma tRNA lacks isopentenyladenosine, or any related hypermodified nucleoside, it appears that the presence of such nucleosides in tRNA is not an absolute requirement for protein synthesis.     

An expanding genetic code

A general method was recently developed that makes it possible to genetically encode unnatural amino acids (UAAs) with diverse physical, chemical or biological properties in Escherichia coli, yeast, and mammalian cells. Over 30 UAAs have been cotranslationally incorporated into proteins with high fidelity and efficiency by means of a unique codon and corresponding tRNA-synthetase pair. A key feature of this methodology is the orthogonality between the new translational components and their endogenous host counterparts. Specifically, the codon for the UAA should not encode a common amino acid; neither the new tRNA nor cognate aminoacyl tRNA synthetase should cross-react with any endogenous tRNA-synthetase pairs; and the new synthetase should recognize only the UAA and not any of the 20 common amino acids. This methodology provides a powerful tool for exploring protein structure and function both in vitro and in vivo, as well as generating proteins with new or enhanced properties.     

Expansion of the genetic code: site-directed p-fluoro-phenylalanine incorporation in Escherichia coli.

Site-directed incorporation of the amino acid analogue p-fluoro-phenylalanine (p-F-Phe) was achieved in Escherichia coli. A yeast suppressor tRNA(Phe)amber/phenylalanyl-tRNA synthetase pair was expressed in an analogue-resistant E. coli strain to direct analogue incorporation at a programmed amber stop codon in the DHFR marker protein. The programmed position was translated to 64-75% as p-F-Phe and the remainder as phenylalanine and lysine. Depending on the expression conditions, the p-F-Phe incorporation was 11-21-fold higher at the programmed position than the background incorporation at phenylalanine codons, showing high specificity of analogue incorporation. Protein expression yields of 8-12 mg/L of culture, corresponding to about two thirds of the expression level of the wild-type DHFR protein, are sufficient to provide fluorinated proteins suitable for 19F-NMR spectroscopy and other sample-intensive methods. The use of a nonessential "21st" tRNA/synthetase pair will permit incorporation of a wide range of analogues, once the synthetase specificity has been modified accordingly.     

Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells

A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.     

Yeast suppressors of UAA and UAG nonsense codons work efficiently in vitro via tRNA

A cell-free protein-synthesizing system, containing an S-100 fraction from yeast, ribosomal subunits from Krebs ascites cells, and ribosome initiation factors from rabbit reticulocytes, translates yeast, adenovirus, and rabbit globin messenger RNAs and the RNA from bacteriophage Qβ. An amber mutation in the Qβ synthetase gene is suppressed in vitro if the S-100 fraction is from yeast strains carrying amber suppressor mutations. Suppressor SUP6-2 gives 16% suppression, and the recessive lethal suppressor RL-1 gives 50% suppression. Extracts from strain FM6, which has the ochre suppressor SUP4-1, give a longer protein product from the normal synthetase gene of Qβ with an efficiency of 63%. This implies that UAA is the terminator for the synthetase gene, and that synthesis of this read through protein can be used as an assay for ochre suppression. Suppression in each of these cases is mediated by tRNA, since purified tRNA is the only fraction from suppressing strains that is required in an otherwise nonsuppressing cell-free system.     

Fluorescent labeling of cell-free synthesized proteins by incorporation of fluorophore-conjugated nonnatural amino acids

Although fluorescent dyes, such as fluorescein derivatives, have bulky and complex structures, nonnatural amino acids carrying these fluorescein derivatives are acceptable by the Escherichia coli ribosome and are useful for the cotranslational fluorescent labeling of cell-free synthesized proteins. Surprisingly, the incorporation efficiency of nonnatural amino acids carrying fluorescein derivatives into translated proteins depends on the source of the translational machinery used in cell-free protein synthesis. That is, whereas the E. coli ribosome efficiently supported the incorporation of nonnatural amino acids carrying fluorescein derivatives into a protein structure, no detectable fluorescent signal was observed from the protein expressed in the eukaryotic cell-free protein synthesis system performed in the presence of fluorescein-conjugated aminoacylated transfer RNA (tRNA).     

Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase

In certain methanogenic archaea a new amino acid, pyrrolysine (Pyl), is inserted at in-frame UAG codons in the mRNAs of some methyltransferases. Pyl is directly acylated onto a suppressor tRNA(Pyl) by pyrrolysyl-tRNA synthetase (PylRS). Due to the lack of a readily available Pyl source, we looked for structural analogues that could be aminoacylated by PylRS onto tRNA(Pyl). We report here the in vitro aminoacylation of tRNA(Pyl) by PylRS with two Pyl analogues: N-epsilon-d-prolyl-l-lysine (d-prolyl-lysine) and N-epsilon-cyclopentyloxycarbonyl-l-lysine (Cyc). Escherichia coli, transformed with the tRNA(Pyl) and PylRS genes, suppressed a lacZ amber mutant dependent on the presence of d-prolyl-lysine or Cyc in the medium, implying that the E. coli translation machinery is able to use Cyc-tRNA(Pyl) and d-prolyl-lysine-tRNA(Pyl) as substrates during protein synthesis. Furthermore, the formation of active beta-galactosidase shows that a specialized mRNA motif is not essential for stop-codon recoding, unlike for selenocysteine incorporation.     

Adding pyrrolysine to the Escherichia coli genetic code

Pyrrolysyl-tRNA synthetase and its cognate suppressor tRNA(Pyl) mediate pyrrolysine (Pyl) insertion at in frame UAG codons. The presence of an RNA hairpin structure named Pyl insertion structure (PYLIS) downstream of the suppression site has been shown to stimulate the insertion of Pyl in archaea. We study here the impact of the presence of PYLIS on the level of Pyl and the Pyl analog N-epsilon-cyclopentyloxycarbonyl-l-lysine (Cyc) incorporation using a quantitative lacZ-luc tandem reporter system in an Escherichia coli context. We show that PYLIS has no effect on the level of neither Pyl nor Cyc incorporation. Exogenously supplying our reporter system with d-ornithine significantly increases suppression efficiency, indicating that d-ornithine is a direct precursor to Pyl.     

High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids

We describe an E. coli-based cell-free system for the production of proteins with a non-natural amino acid (nnAA) incorporated site-specifically (modified protein). The mutant Methanococcus jannaschii tyrosyl-tRNA synthetase (mTyrRS) and tRNA(Tyr) pair were used as orthogonal elements. The mTyrRS experienced proteolysis and modified protein yields improved with higher synthetase addition (200-300 microg/mL). Product yields were also improved by increasing levels of total protein to 20 mg protein/mL and available vesicle surface area to 0.5 m(2)/mL. This new E. coli-based cell-free procedure produced up to 400 microg/mL of eCAT109pAz, 660 microg/mL of eDHFR10pAz, and 210 microg/mL of mDHFR31pAz with p-azido-L-phenylalanine (pAz) incorporated site-specifically at the amber nonsense codon. O-methyl-L-tyrosine and p-acetyl-L-phenylalanine were incorporated by similar protocols. The desired specificity for incorporation of the nnAA by the cell-free system was confirmed. Additionally, the modified proteins were enzymatically active and reactive for copper(I)-catalyzed (3 + 2) cycloadditions (click chemistry).     

In vitro selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an Escherichia coli cell-free translation system

Position-specific incorporation of non-natural amino acids into proteins is a useful technique in protein engineering. In this study, we established a novel selection system to obtain tRNAs that show high decoding activity, from a tRNA library in a cell-free translation system to improve the efficiency of incorporation of non-natural amino acids into proteins. In this system, a puromycin-tRNA conjugate, in which the 3'-terminal A unit was replaced by puromycin, was used. The puromycin-tRNA conjugate was fused to a C-terminus of streptavidin through the puromycin moiety in the ribosome. The streptavidin-puromycin-tRNA fusion molecule was collected and brought to the next round after amplification of the tRNA sequence. We applied this system to select efficient frameshift suppressor tRNAs from a tRNA library with a randomly mutated anticodon loop derived from yeast tRNA CCCG Phe. After three rounds of the selection, we obtained novel frameshift suppressor tRNAs which had high decoding activity and good orthogonality against endogenous aminoacyl-tRNA synthetases. These results demonstrate that the in vitro selection system developed here is useful to obtain highly active tRNAs for the incorporation of non-natural amino acid from a tRNA library.     

A cell-free protein synthesis system as an investigational tool for the translation stop processes

Using Escherichia coli cell-free protein synthesis system and aminoacylated amber suppressor tRNA, we successfully inserted an unnatural amino acid S-(2-nitrobenzyl)cysteine into human erythropoietin. Three different types of translation stop suppression were observed and each of the three types was easily discerned with SDS-PAGE. Optimal conditions were established for correct stop and programmed suppressions. Since this system differentiates proteins produced by misreading of codons from those produced by programmed suppression, we conclude that this cell-free translation system that we describe in this paper will be of a great use for future investigations on translation stop processes.     

A Robust Platform for Unnatural Amino Acid Mutagenesis in E. coli Using the Bacterial Tryptophanyl-tRNA synthetase/tRNA pair

We report the development of a robust user-friendly Escherichia coli (E. coli) expression system, derived from the BL21(DE3) strain, for site-specifically incorporating unnatural amino acids (UAAs) into proteins using engineered E. coli tryptophanyl-tRNA synthetase (EcTrpRS)-tRNA^Trp pairs. This was made possible by functionally replacing the endogenous EcTrpRS-tRNA^Trp pair in BL21(DE3) E. coli with an orthogonal counterpart from Saccharomyces cerevisiae, and reintroducing it into the resulting altered translational machinery tryptophanyl (ATMW-BL21) E. coli strain as an orthogonal nonsense suppressor. The resulting expression system benefits from the favorable characteristics of BL21(DE3) as an expression host, and is compatible with the broadly used T7-driven recombinant expression system. Furthermore, the vector expressing the nonsense-suppressing engineered EcTrpRS-tRNA^Trp pair was systematically optimized to significantly enhance the incorporation efficiency of various tryptophan analogs. Together, the improved strain and the optimized suppressor plasmids enable efficient UAA incorporation (up to 65% of wild-type levels) into several different proteins. This robust and user-friendly platform will significantly expand the scope of the genetically encoded tryptophan-derived UAAs.     

Construction of Escherichia coli amber suppressor tRNA genes. III. Determination of tRNA specificity

Using synthetic oligonucleotides, we have constructed a collection of Escherichia coli amber suppressor tRNA genes. In order to determine their specificities, these tRNAs were each used to suppress an amber (UAG) nonsense mutation in the E. coli dihydrofolate reductase gene fol. The mutant proteins were purified and subjected to N-terminal sequence analysis to determine which amino acid had been inserted by the suppressor tRNAs at the position of the amber codon. The suppressors can be classified into three groups on the basis of the protein sequence information. Class I suppressors, tRNA(CUAAla2), tRNA(CUAGly1), tRNA(CUAHisA), tRNA(CUALys) and tRNA(CUAProH), inserted the predicted amino acid. The class II suppressors, tRNA(CUAGluA), tRNA(CUAGly2) and tRNA(CUAIle1) were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase. The class III suppressors, tRNA(CUAArg), tRNA(CUAAspM), tRNA(CUAIle2), tRNA(CUAThr2), tRNA(CUAMet(m)) and tRNA(CUAVal) inserted predominantly lysine.     

A bacterial amber suppressor in Saccharomyces cerevisiae is selectively recognized by a bacterial aminoacyl-tRNA synthetase.

Little is known about the conservation of determinants for the identities of tRNAs between organisms. We showed previously that Escherichia coli tyrosine tRNA synthetase can charge the Saccharomyces cerevisiae mitochondrial tyrosine tRNA in vivo, even though there are substantial sequence differences between the yeast mitochondrial and bacterial tRNAs. The S. cerevisiae cytoplasmic tyrosine tRNA differs in sequence from both its yeast mitochondrial and E. coli counterparts. To test whether the yeast cytoplasmic tyrosyl-tRNA synthetase recognizes the E. coli tRNA, we expressed various amounts of an E. coli tyrosine tRNA amber suppressor in S. cerevisiae. The bacterial tRNA did not suppress any of three yeast amber alleles, suggesting that the yeast enzymes retain high specificity in vivo for their homologous tRNAs. Moreover, the nucleotides in the sequence of the E. coli suppressor that are not shared with the yeast cytoplasmic tyrosine tRNA do not create determinants which are efficiently recognized by other yeast charging enzymes. Therefore, at least some of the determinants that influence in vivo recognition of the tyrosine tRNA are specific to the cell compartment and organism. In contrast, expression of the cognate bacterial tyrosyl-tRNA synthetase together with the bacterial suppressor tRNA led to suppression of all three amber alleles. The bacterial enzyme recognized its substrate in vivo, even when the amount of bacterial tRNA was less than about 0.05% of that of the total cytoplasmic tRNA.     

Rewiring translation - Genetic code expansion and its applications

With few minor variations, the genetic code is universal to all forms of life on our planet. It is difficult to imagine that one day organisms might exist that use an entirely different code to translate the information of their genome. Recent developments in the field of synthetic biology, however, have opened the gate to their creation. The genetic code of several organisms has been expanded by the heterologous expression of evolved aminoacyl-tRNA synthetase/tRNA(CUA) pairs that mediate the incorporation of unnatural amino acids in response to amber codons. These UAAs introduce exciting new features into proteins, such as spectroscopic probes, UV-inducible crosslinkers, and functional groups for bioorthogonal conjugations or posttranslational modifications. Orthogonal ribosomes provide a parallel translational machinery in Escherichia coli that has lost its evolutionary constraints. Evolved variants of these ribosomes translate amber or quadruplet codons with massively enhanced efficiency. Here, I review these recent developments emphasizing their tremendous potential to facilitate biochemical and cell biological studies.     

Site-specific incorporation of unnatural amino acids into urate oxidase in Escherichia coli

Urate oxidase catalyzes the oxidation of uric acid with poor solubility to produce 5-hydroxyisourate and allantoin. Since allantoin is excreted in vivo, urate oxidase has the potential to be a therapeutic target for the treatment of gout. However, its severe immunogenicity limits its clinical application. Furthermore, studies on the structure-function relationships of urate oxidase have proven difficult. We developed a method for genetically incorporating p-azido-L-phenylalanine into target protein in Escherichia coli in a site-specific manner utilizing a tyrosyl suppressor tRNA/aminoacyl-tRNA synthetase system. We substituted p-azido-L-phenylalanine for Phe(170) or Phe(281) in urate oxidase. The products were purified and their enzyme activities were analyzed. In addition, we optimized the system by adding a "Shine-Dalgarno (SD) sequence" and tandem suppressor tRNA. This method has the benefit of site-specifically modifying urate oxidase with homogeneous glycosyl and PEG derivates, which can provide new insights into structure-function relationships and improve pharmacological properties of urate oxidase.