Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate.

BACKGROUND: Angiotensinogen (ANG) is a macromolecular precursor of angiotensin, which regulates blood pressure and electrolyte balance. ANG is specifically cleaved by renin, an aspartic protease, to initiate the angiotensin-processing cascade. Ovine ANG (oANG) from sheep plasma has been shown to be a better substrate for human renin, and it has been used in clinical renin assays. To expand the availability of oANG, we aimed to produce milligram levels of recombinant oANG using an Escherichia coli expression system. RESULTS: When recombinant oANG was expressed from a T7 promoter in various E. coli strains at 37 °C, it accumulated in the insoluble fraction. However, by expressing oANG at 37 °C from a tac promoter, which has weaker transcriptional activity than a T7 promoter, we significantly elevated the ratio of soluble to insoluble recombinant oANG. Using a novel culturing system and auto-induction culture medium, we purified tac-expressed recombinant oANG to homogeneity, with a yield of 4.0 mg per liter of culture. Based on size-exclusion gel filtration analysis and dynamic light scattering analysis, the resulting purified oANG is a monomer in solution. The circular dichroism spectrum of E. coli-expressed recombinant oANG was similar to that of oANG expressed in CHO cells. Differential scanning fluorimetry showed that both preparations undergo a two-state transition during thermal denaturation, and the melting temperatures of recombinant oANG expressed in E. coli and CHO cells were 49.4 ± 0.16 °C and 51.6 ± 0.19 °C, respectively. The K(m) values of both oANG preparations were similar; the k(cat) value of E. coli-expressed recombinant oANG was slightly higher than that of CHO-expressed oANG. CONCLUSIONS: Recombinant oANG expressed in E. coli functions as a human renin substrate. This study presents an E. coli-based system for the rapid production of milligram quantities of a human renin substrate, which will be useful for both fundamental and clinical studies on renin and hypertension.

Correlation between the stability of tRNA tertiary structure and the catalytic efficiency of a tRNA-modifying enzyme, archaeal tRNA-guanine transglycosylase.

In many archaeal tRNAs, archaeosine is found at position 15. During archaeosine biosynthesis, archaeal tRNA-guanine transglycosylase (ArcTGT) first replaces the guanine base at position 15 with 7-cyano-7-deazaguanine (preQ0). In this study, we investigated whether modified nucleosides in tRNA substrates would affect ArcTGT incorporation of preQ0. We prepared a series of hypomodified tRNAs(Ser)(GGA) from Escherichia coli strains lacking each tRNA-modifying enzyme. Measurement of ArcTGT kinetic parameters with the various tRNAs(Ser)(GGA) as substrates showed that the Km decreased due to the lack of modified nucleosides. The tRNAs(Ser)(GGA) melting profiles resulted in experimental evidence showing that each modified nucleoside in tRNA(Ser)(GGA) enhanced tRNA stability. Furthermore, the ArcTGT K(m) strongly correlated with the melting temperature (T(m)), suggesting that the unstable tRNA containing fewer modified nucleosides served as a better ArcTGT substrate. These results show that preQ0 incorporation into tRNA by ArcTGT takes place early in the archaeal tRNA modification process.

Protein fishing using magnetic nanobeads containing calmodulin site-specifically immobilized via an azido group.

We developed a novel method for capturing proteins that interact with a target protein. This method utilizes a protein containing a site-specifically incorporated 3-azidotyrosine (N3-Y) and FG beads for immobilization of the protein via an azido group. Using calmodulin (CaM) as the target protein, we introduced N3-Y at position 72 and conjugated it to FG beads by copper-free click chemistry. From the Ca(2+)/CaM-binding proteins captured from mouse brain cell lysate and analysis by mass spectrometry, we identified six proteins: alpha-enolase (ENOA), glucose-6-phosphate isomerase (GPI), annexin A5 (ANXA5), malate dehydrogenase 1 (MDH1), glyceraldehyde-3-phosphate dehydrogenase and the well-known CaM-binding protein phosphoglycerate kinase 1 (PGK1). The presence of photo-crosslinking products via N3-Y for all the captured proteins except GPI indicated that they bound directly to CaM. In this study, ENOA, ANXA5 and MDH1 were identified as novel CaM-binding proteins, and PGK1 was bound to Ca(2+)/CaM and also Ca(2+)-free CaM. This method should prove useful for capturing novel interacting proteins and serve as a useful tool for proteomic analyses.

A simple system for expression of proteins containing 3-azidotyrosine at a pre-determined site in Escherichia coli.

We developed an efficient method for introduction of 3-azidotyrosine (N(3)-Y) into proteins in Escherichia coli cells. We constructed a plasmid that is adaptable for the constitutive expression of both Methanosarcina acetivorans tyrosyl-tRNA synthetase (TyrRS) and tRNA(()(CUA)), and made an orthogonal tRNA((CUA)) that is recognized as a substrate only by the archaeal TyrRS. Random mutations were introduced into M. acetivorans TyrRS around the tyrosine binding pocket, and a TyrRS mutant recognizing N(3)-Y was selected. We then expressed rat calmodulin (CaM) containing N(3)-Y, using the CaM gene with an amber codon at position 80. Mass analyses confirmed production of CaM containing N(3)-Y, but a significant amount of CaM containing 3-aminotyrosine was also detected. To more efficiently express CaM containing N(3)-Y, we added an arabinose-inducible gene for the mutant TyrRS to the plasmid carrying the mutant TyrRS/tRNA(()(CUA)) gene. Although the yields of full-length CaM increased ~3-fold, the ratio of N(3)-Y introduction was not significantly improved. Following screening for a suitable host cell, we found that CaM expressed in E. coli SHuffle (K-12) had 97% N(3)-Y at the pre-determined site. Finally, we obtained up to 2 mg of CaM containing N(3)-Y per 100 ml of culture media, sufficient for use in various proteomics experiments, including photo-crosslinking.

Involvement of plasmin-mediated extracellular activation of progalanin in angiogenesis.

Progalanin is released from the small cell lung carcinoma line SBC-3A and converted to its active form by plasmin. To elucidate the role of progalanin activation in the extracellular compartment, matrix metalloproteinase (MMP) activity was studied in SBC-3A cells treated with progalanin siRNA, and angiogenesis was measured in tumor tissue originating from SBC-3A cell transplantation into mice. Progalanin siRNA caused downregulation of progalanin expression for approximately 8 days. MMP activity and angiogenesis were reduced in tumors induced by transplantation of progalanin siRNA-treated SBC-3A cells. In contrast, MMP-9 and MMP-2 activity and angiogenesis increased in tumors originating from progalanin siRNA-treated SBC-3A cells in the presence of galanin and progalanin. Furthermore, injection of tranexamic acid, a plasmin inhibitor, more markedly reduced MMP-9 and MMP-2 activity and angiogenesis in tumors originating from progalanin siRNA-treated SBC-3A cells and in tumor tissue originating from progalanin siRNA-treated SBC-3A cells in the presence of progalanin. The reduction of MMP-9 and MMP-2 activity with tranexamic acid was restored by galanin, but not by progalanin. Moreover, tranexamic acid reduced angiogenesis in control siRNA-treated SBC-3A cells. These results suggest that the activation of progalanin by plasmin in the extracellular compartment was involved in MMP-9 and MMP-2 activation and in angiogenesis in tumor tissue.

Purification and comparison of native and recombinant tRNA-guanine transglycosylases from Methanosarcina acetivorans.

Many archaeal tRNAs have archaeosine (G(+)) at position 15 in the D-loop and this is thought to strengthen the tertiary interaction with C48 in the V-loop. In the first step of G(+) biosynthesis, archaeosine tRNA-guanine transglycosylase (ArcTGT)(1) catalyzes the base exchange reaction from guanine to 7-cyano-7-deazaguanine (preQ(0)). ArcTGT is classified into full-size or split types, according to databases of genomic information. Although the full-size type forms a homodimeric structure, the split type has been assumed to form a heterotetrameric structure, consisting of two kinds of peptide. However, there has been no definitive evidence for this presented to date. Here, we show that native ArcTGT could be isolated from Methanosarcina acetivorans and two peptides formed a robust complex in cells. Consequently, the two peptides function as actual subunits of ArcTGT. We also overexpressed recombinant ArcTGT in Escherichia coli cells. Product was successfully obtained by co-overexpression of the two subunits but one subunit alone was not adequately expressed in soluble fractions. This result suggests that interaction between the two subunits may contribute to the conformational stability of split ArcTGT. The values of the kinetic parameters for the recombinant and native ArcTGT were closely similar. Moreover, tRNA transcript with preQ(0) at position 15 was successfully prepared using the recombinant ArcTGT. This tRNA transcript is expected to be useful as a substrate for studies seeking the enzymes responsible for G(+) biosynthesis.

Site-specific attachment of a protein to a carbon nanotube end without loss of protein function.

Establishing a nanobiohybrid device largely relies on the availability of various bioconjugation procedures which allow coupling of biomolecules and inorganic materials. Especially, site-specific coupling of a protein to nanomaterials is highly useful and significant, since it can avoid adversely affecting the protein's function. In this study, we demonstrated a covalent coupling of a protein of interest to the end of carbon nanotubes without affecting protein's function. A modified Staudinger-Bertozzi ligation was utilized to couple a carbon nanotube end with an azide group which is site-specifically incorporated into a protein of interest. We demonstrated that Ca(2+)-sensor protein, calmodulin, can be attached to the end of the nanotubes without affecting the ability to bind to the substrate in a calcium-dependent manner. This procedure can be applied not only to nanotubes, but also to other nanomaterials, and therefore provides a fundamental technique for well-controlled protein conjugation.

Activation of large form galanin-LI by extracellular processing in small cell lung carcinoma tissue.

Galanin is a neuropeptide that is widely distributed in the central and peripheral nervous systems. Some small cell lung carcinoma (SCLC) cell lines such as SBC-3A release only the high-molecular-mass form, with lower molecular mass forms being undetectable. To investigate the mechanism of processing of progalanin to active peptide, we studied galanin-LI in both the culture media of SBC-3A cells and in extracts from in vivo mouse SBC-3A tumors. SBC-3A cells were found to release high molecular mass galanin, but did not release active peptides. In contrast, tumor extract contained both high-molecular-mass galanin, and a cleaved lower-molecular-mass form of the peptide (8, 5 and 2 kDa). The lower-molecular-mass peptide was identified as galanin(1-20) by MALDI-TOF mass spectrometry. We then looked at MMP-2 and MMP-9 release from SBC-3A cells and tumor tissue treated with galanin and progalanin, as revealed by gelatin zymography. Galanin elicited pro-MMP-2 and pro-MMP-9 release from SBC-3A cells and tumor tissue; however, recombinant progalanin induced pro-MMP-2 and pro-MMP-9 release from tumor tissue only. This study has shown that the galanin-LI released from SCLC SBC-3A cells consisted of the high-molecular-mass peptide form, and was processed extracellularly to galanin(1-20). Furthermore, galanin was seen to induce pro-MMP-2 and pro-MMP-9 release from SBC-3A cells.

Incorporation of 3-azidotyrosine into proteins through engineering yeast tyrosyl-tRNA synthetase and its application to site-selective protein modification.

An efficient method for site-selective introduction of 3-azidotyrosine into proteins has been developed. This method utilizes the yeast amber suppressor tRNA(Tyr)/mutant tyrosyl-tRNA synthetase (Y43G) pair as the carrier of 3-azidotyrosine in an Escherichia coli cell-free translation system. Using rat calmodulin (CaM) as a model protein, we prepared an unnatural CaM molecule carrying a 3-azidotyrosine residue at the predetermined position 80. The synthesized CaM containing 3-azidotyrosine was site-specifically modified via azido group with a fluorescent alkyne derivative by click chemistry. This method will be useful to prepare not only a cross-linkable protein containing 3-azidotyrosine but also a fluorescent protein with a single fluorophore to facilitate the elucidation of molecular mechanisms of protein functions and protein-to-protein networks.

Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion.

Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase-tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)-tRNA(Tyr) pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS-tRNA(Tyr) pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNA(Tyr). The endogenous TyrRS and tRNA(Tyr) genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS-tRNA(Tyr) pair. In this engineered strain, 3-iodo-L-tyrosine and 3-azido-L-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-L-tyrosine and was also found to recognize 3-azido-L-tyrosine. The structural basis for the 3-azido-L-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.

Optimization of the hybridization-based method for purification of thermostable tRNAs in the presence of tetraalkylammonium salts.

We found that both tetramethylammonium chloride (TMA-Cl) and tetra-ethylammonium chloride (TEA-Cl), which are used as monovalent cations for northern hybridization, drastically destabilized the tertiary structures of tRNAs and enhanced the formation of tRNA*oligoDNA hybrids. These effects are of great advantage for the hybridization-based method for purification of specific tRNAs from unfractionated tRNA mixtures through the use of an immobilized oligoDNA complementary to the target tRNA. Replacement of NaCl by TMA-Cl or TEA-Cl in the hybridization buffer greatly improved the recovery of a specific tRNA, even from unfractionated tRNAs derived from a thermophile. Since TEA-Cl destabilized tRNAs more strongly than TMA-Cl, it was necessary to lower the hybridization temperature at the sacrifice of the purity of the recovered tRNA when using TEA-Cl. Therefore, we propose two alternative protocols, depending on the desired properties of the tRNA to be purified. When the total recovery of the tRNA is important, hybridization should be carried out in the presence of TEA-Cl. However, if the purity of the recovered tRNA is important, TMA-Cl should be used for the hybridization. In principle, this procedure for tRNA purification should be applicable to any small-size RNA whose gene sequence is already known.

Preparation of an ochre suppressor tRNA recognizing exclusively UAA codon by using the molecular surgery technique.

In order to create an ochre suppressor tRNA which exclusively recognizes UAA codon, we replaced the G34 at the first position of yeast tRNA(Tyr)[GPsiA] anticodon with pseudouridine34 (Psi34) by using the molecular surgery technique. This tRNA(Tyr)[PsiPsiA] recognized only the UAA codon as expectedly, but tRNA(Tyr)[UPsiA] made as a control also behaved similarly. This result may suggest that U34 must be somehow modified to facilitate the wobble-pairing to G at the third position of codon.

Detection of enzymatic activities related to the tRNA splicing pathway in Methanosarcina acetivorans cell extract.

At least two separate enzymes, an endonuclease and a ligase, are thought to be involved in the tRNA splicing pathway. The yeast and archaeal endonucleases acting in the first step of tRNA splicing commonly produce 2', 3'-cyclic phosphate and 5' hydroxy group at the exon-intron borders. Despite this similarity in the first step of tRNA splicing, the subsequent mechanism of archaeal splicing pathway has not been elucidated yet. We have been searching for the archaeal ligase activity from Methanosarcina acetivorans. Here, we report the distinct activity of a splicing endonuclease detected in its cell extract.

Aquifex aeolicus tRNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes transfer of methyl groups not only to guanine 26 but also to guanine 27 in tRNA.

Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m(2)(2)G26) in tRNA. In the reaction, N2-guanine at position 26 (m(2)G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNA(Cys) has an m(2)(2)G26m(2)G27 or m(2)(2)G26m(2)(2)G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m2G26 formation is faster than the m(2)G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme.

Structural basis for recognition of cognate tRNA by tyrosyl-tRNA synthetase from three kingdoms.

The specific aminoacylation of tRNA by tyrosyl-tRNA synthetases (TyrRSs) relies on the identity determinants in the cognate tRNA(Tyr)s. We have determined the crystal structure of Saccharomyces cerevisiae TyrRS (SceTyrRS) complexed with a Tyr-AMP analog and the native tRNA(Tyr)(GPsiA). Structural information for TyrRS-tRNA(Tyr) complexes is now full-line for three kingdoms. Because the archaeal/eukaryotic TyrRSs-tRNA(Tyr)s pairs do not cross-react with their bacterial counterparts, the recognition modes of the identity determinants by the archaeal/eukaryotic TyrRSs were expected to be similar to each other but different from that by the bacterial TyrRSs. Interestingly, however, the tRNA(Tyr) recognition modes of SceTyrRS have both similarities and differences compared with those in the archaeal TyrRS: the recognition of the C1-G72 base pair by SceTyrRS is similar to that by the archaeal TyrRS, whereas the recognition of the A73 by SceTyrRS is different from that by the archaeal TyrRS but similar to that by the bacterial TyrRS. Thus, the lack of cross-reactivity between archaeal/eukaryotic and bacterial TyrRS-tRNA(Tyr) pairs most probably lies in the different sequence of the last base pair of the acceptor stem (C1-G72 vs G1-C72) of tRNA(Tyr). On the other hand, the recognition mode of Tyr-AMP is conserved among the TyrRSs from the three kingdoms.

Extremely thermophilic translation system in the common ancestor commonote: ancestral mutants of Glycyl-tRNA synthetase from the extreme thermophile Thermus thermophilus.

Based on phylogenetic analysis of 16 S and 18 S rRNAs, the common ancestor of all organisms (Commonote) was proposed to be hyperthermophilic. We have previously tested this hypothesis using enzymes with ancestral residues that are inferred by molecular phylogenetic analysis. The ancestral mutant enzymes involved in metabolic systems show higher thermal stability than wild-type enzymes, consistent with the hyperthermophile common ancestor hypothesis. Here, we have extended the experiments to include an enzyme of the translation system, glycyl-tRNA synthetase (GlyRS). The translation system often shows a phylogenetic tree that is similar to the rRNA tree. Thus, it is likely that the tree represents the evolutionary route of the organisms. The maximum-likelihood tree of alpha(2) type GlyRS was constructed. From this analysis the ancestral sequence of GlyRS was deduced and individual or pairs of ancestral residues were introduced into Thermus thermophilus GlyRS. The ancestral mutants were expressed in Escherichia coli, purified and activity measured. The thermostability of eight mutated proteins was evaluated by CD (circular dichroism) measurements. Six mutants showed higher thermostability than wild-type enzyme and seven mutants showed higher activity than wild-type enzyme at 70 degrees C, suggesting an extremely thermophilic translation system in the common ancestor Commonote.

Detection of structural changes in a cofactor binding protein by using a wheat germ cell-free protein synthesis system coupled with unnatural amino acid probing.

A cell-free protein synthesis system is a powerful tool with which unnatural amino acids can be introduced into polypeptide chains. Here, the authors describe unnatural amino acid probing in a wheat germ cell-free translation system as a method for detecting the structural changes that occur in a cofactor binding protein on a conversion of the protein from an apo-form to a holo-form. The authors selected the FMN-binding protein from Desulfovibrio vulgaris as a model protein. The apo-form of the protein was synthesized efficiently in the absence of FMN. The purified apo-form could be correctly converted to the holo-form. Thus, the system could synthesize the active apo-form. Gel filtration chromatography, analytical ultracentrifugation, and circular dichroism-spectra studies suggested that the FMN-binding site of the apo-form is open as compared with the holo-form. To confirm this idea, the unnatural amino acid probing was performed by incorporating 3-azido-L-tyrosine at the Tyr35 residue in the FMN-binding site. The authors optimized three steps in their system. The introduced 3-azido-L-tyrosine residue was subjected to specific chemical modification by a fluorescein-triarylphosphine derivative. The initial velocity of the apo-form reaction was 20 fold faster than that of the holo-form, demonstrating that the Tyr35 residue in the apo-form is open to solvent.

Site-selective post-translational modification of proteins using an unnatural amino acid, 3-azidotyrosine.

An efficient method for site-selective modification of proteins using an unnatural amino acid, 3-azidotyrosine has been developed. This method utilizes the yeast amber suppressor tRNA(Tyr)/mutated tyrosyl-tRNA synthetase pair as a carrier of 3-azidotyrosine in an Escherichia coli cell-free translation system, and triarylphosphine derivatives for specific modification of the azido group. Using rat calmodulin (CaM) as a model protein, we prepared several unnatural CaM molecules, each carrying an azidotyrosine at predetermined positions 72, 78, 80 or 100, respectively. Post-translational modification of these proteins with a conjugate compound of triarylphosphine and biotin produced site-selectively biotinylated CaM molecules. Reaction efficiency was similar among these proteins irrespective of the position of introduction, and site-specificity of biotinylation was confirmed using mass spectrometry. In addition, CBP-binding activity of the biotinylated CaMs was confirmed to be similar to that of wild-type CaM. This method is intrinsically versatile in that it should be easily applicable to introducing any other desirable compounds (e.g., probes and cross-linkers) into selected sites of proteins as far as appropriate derivative compounds of triarylphosphine could be chemically synthesized. Elucidation of molecular mechanisms of protein functions and protein-to-protein networks will be greatly facilitated by making use of these site-selectively modified proteins.

A base pair at the bottom of the anticodon stem is reciprocally preferred for discrimination of cognate tRNAs by Escherichia coli lysyl- and glutaminyl-tRNA synthetases.

Although the yeast amber suppressor tRNA(Tyr) is a good candidate for a carrier of unnatural amino acids into proteins, slight misacylation with lysine was found to occur in an Escherichia coli protein synthesis system. Although it was possible to restrain the mislysylation by genetically engineering the anticodon stem region of the amber suppressor tRNA(Tyr), the mutant tRNA showing the lowest acceptance of lysine was found to accept a trace level of glutamine instead. Moreover, the glutamine-acceptance of various tRNA(Tyr) transcripts substituted at the anticodon stem region varied in reverse proportion to the lysine-acceptance, similar to a 'seesaw'. The introduction of a C31-G39 base pair at the site was most effective for decreasing the lysine-acceptance and increasing the glutamine-acceptance. When the same substitution was introduced into E.coli tRNA(Lys) transcripts, the lysine-accepting activity was decreased by 100-fold and faint acceptance of glutamine was observed. These results may support the idea that there are some structural element(s) in the anticodon stem of tRNA, which are not shared by aminoacyl-tRNA synthetases that have similar recognition sites in the anticodon, such as E.coli lysyl- and glutaminyl-tRNA synthetases.

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.