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Engineering G-protein-coupled receptors (GPCRs) for improved stability or altered function is of great interest, as GPCRs consist of the largest protein family, are involved in many important signaling pathways, and thus, are one of the major drug targets. Here, we report the development of a high-throughput screening method for GPCRs using a reconstituted in vitro transcription-translation (IVTT) system. Human endothelin receptor type-B (ETBR), a class A GPCR that binds endothelin-1 (ET-1), a 21-residue peptide hormone, was synthesized in the presence of nanodisc (ND) composed of a phospholipid, 1-palmitoyl-2-oleoyl

We performed metadynamics molecular dynamics simulations to reveal mechanism of antigen-dependent fluorescence response observed for site-specifically fluorescent-labeled single-chain antibody against c-Myc peptide antigen. We found that V H and V L bind with each other only when the antigen exists and that the fluorophore labeled at the N-terminus of V H interacts with Trp103 most stably. These results support the mechanism proposed from previous experiments: In the absence of antigen, Trp residues are partially exposed at the interface of V H and quench the fluorophore. In the presence of antigen, the Trp residues are buried between V H and V L , and the quenching is eliminated.

Physical methodologies such as electroporation and the gene-gun technology have been widely used for transfection; however, their applicability is limited because they lead to cell damage and low cell viability. Therefore, to address these limitations we developed a new freeze concentration-based gene transfection system that provides enhanced in vitro gene delivery compared to that provided by the commercially available systems. The system employs a facile freeze concentration step, whereby cells are simply frozen to very low temperatures in the presence of polymer-pDNA complexes. As part of system development, we also synthesized a low toxicity polyethyleneimine (PEI)-based polyampholyte prepared through succinylation with butylsuccinic anhydride. In aqueous solution, this modified polyampholyte self-assembles to form small (20 nm diameter), positively charged (net surface charge of 35 mV), nanoparticles through a combination of hydrophobic and electrostatic interactions. Agarose gel electrophoresis analysis indicated that the polyampholyte nanoparticle was able to form a complex with pDNA that provided stability against nuclease degradation. Using transfection of HEK-293T cells, we demonstrated that a combination of polyampholyte: pDNA, at an appropriate ratio, and the freeze concentration method resulted in significant enhancement of GFP and luciferase expression compared to commercially available carriers. Endosomal escape of pDNA was also found to be increased when using the modified polyampholyte compared to branched PEI. This study suggests that the efficient combination of freeze concentration and the modified polyampholyte described here has great potential for in vitro gene therapy.

The bacterial cell surface structure has important roles for various cellular functions. However, research on reconstituting bacterial cell surface structures is limited. This study aimed to bottom-up create a cell-sized liposome covered with AtaA, the adhesive bacterionanofiber protein localized on the cell surface of Acinetobacter sp. Tol 5, without the use of the protein secretion and assembly machineries. Liposomes containing a benzylguanine derivative-modified phospholipid were decorated with a truncated AtaA protein fused to a SNAP-tag expressed in a soluble fraction in Escherichia coli. The obtained liposome showed a similar surface structure and function to that of native Tol 5 cells and adhered to both hydrophobic and hydrophilic solid surfaces. Furthermore, this artificial cell was able to drive an enzymatic reaction in the adhesive state. The developed artificial cellular system will allow for analysis of not only AtaA, but also other cell surface proteins under a cell-mimicking environment. In addition, AtaA-decorated artificial cells may inspire the development of biotechnological applications that require immobilization of cells onto a variety of solid surfaces, in particular, in environments where the use of genetically modified organisms is prohibited.

Fluorescence ratio probes are useful tools for quantitative detection of target molecules even if the concentration of probe molecules is unknown. However, a general and widely applicable method fo...

In the present study, a novel molecular biosensor system model was designed by using a couple of the fluorescent unnatural mutant streptavidin and the carbazole-labeled biotin. BODIPY-FL-aminophenylalanine (BFLAF), a fluorescent unnatural amino acid was position-specifically incorporated into Trp120 position of streptavidin by four-base codon method. On the other hand, carbazole-labeled biotin was synthesized as a quencher for the fluorescent Trp120BFLAF mutant streptavidin. The fluorescence of fluorescent Trp120BFLAF mutant streptavidin was decreased as we expected when carbazole-labeled biotin was added into the mutant streptavidin solution. Furthermore, the fluorescence decrease of Trp120BFLAF mutant streptavidin with carbazole-labeled biotin (100 nM) was recovered by the competitive addition of natural biotin. This result demonstrated that by measuring the fluorescence quenching and recovery, a couple of the fluorescent Trp120BFLAF mutant streptavidin and the carbazole-labeled biotin were successfully applicable for quantification of free biotin as a molecular biosensor system. Copyright © 2016 John Wiley & Sons, Ltd.

Z-Lysine (ZLys) is a lysine derivative with a benzyloxycarbonyl group linked to the ε-nitrogen. It has been genetically encoded with the UAG stop codon, using the pair of an engineered variant of pyrrolysyl-tRNA synthetase (PylRS) and tRNA(Pyl). In the present study, we designed a novel Z-lysine derivative (AmAzZLys), which is doubly functionalized with amino and azido substituents at the meta positions of the benzyl moiety, and demonstrated its applicability for creating protein conjugates. AmAzZLys was incorporated into proteins in Escherichia coli, by using the ZLys-specific PylRS variant. AmAzZLys was then site-specifically incorporated into a camelid single-domain antibody specific to the epidermal growth factor receptor (EGFR). A one-pot reaction demonstrated that the phenyl amine and azide were efficiently linked to the 5 kDa polyethylene glycol and a fluorescent probe, respectively, through specific bio-orthogonal chemistry. The antibody was then tested for the ability to form a photo-cross-link between its phenylazide moiety and the antigen, while the amino group on the same ring was used for chemical labeling. When incorporated at a selected position in the antibody and exposed to 365 nm light, AmAzZLys formed a covalent bond with the EGFR ectodomain, with the phenylamine moiety labeled fluorescently prior to the reaction. The present results illuminated the versatility of the ZLys scaffold, which can accommodate multiple reactive groups useful for protein conjugation.

Membranes of living cells possess asymmetry. The inner and outer leaflets of the membrane consist of different phospholipid compositions, which are known to affect the function of membrane proteins, and the loss of the asymmetry has been reported to lead to cell apoptosis. In addition, different proteins are found on the inner and outer leaflets of the membrane, and they are essential for various biochemical reactions, including those related to signal transduction and cell morphology. While in vitro lipid bilayer reconstitution with asymmetric phospholipid compositions has been reported, the reconstitution of lipid bilayer where different proteins are localized in the inner and outer leaflet, thereby enables asymmetric protein localizations, has remained difficult. Herein, we developed a simple method to achieve this asymmetry using an in vitro transcription–translation system (IVTT). The method used a benzylguanine (BG) derivative-modified phospholipid, which forms a covalent bond with a snap-tag sequence. We show that purified snap-tagged protein can be localized to the cell-sized liposome surface via an interaction between BG and the snap-tag. We then show that IVTT-synthesized proteins can be located at the lipid membrane and that different proteins can be asymmetrically localized on the outer and inner leaflets of liposomes.

Molecular biosensors represent a powerful form of biotechnology. In this study, a novel molecular biosensor model was designed based on fluorescence resonance energy transfer (FRET) that was carried out by using a couple composed of a genetically engineered fluorescent mutant binding protein and fluorophore-labeled ligand analogue. The pair of streptavidin and biotin was used as a model here. Trp120BFLAF mutant streptavidin (a fluorescent unnatural amino acid named BODIPY-FL-aminophenylalanine (BFLAF)) was position-specifically incorporated into the Trp120 position of streptavidin by a four-base codon method. The FRET occurred when the Alexa546-biotin was added into the Trp120 BFLAF mutant streptavidin. Furthermore, the FRET between Trp120BFLAF mutant streptavidin and Alexa546-biotin decreased by the competitive addition of natural biotin. This result demonstrated that by measuring the FRET, a couple composed of a genetically engineered fluorescent mutant binding protein (Trp120BFLAF mutant streptavidin) and fluorophore-labeled ligand analogue (Alexa546-biotin) was applicable for the quantification of the small ligand (free biotin) as a molecular biosensor system.

Structural characterization of fully unfolded proteins is essential for understanding not only protein-folding mechanisms, but also the structures of intrinsically disordered proteins. Because an unfolded protein can assume all possible conformations, statistical descriptions of its structure are most appropriate. For this purpose, we applied Forster resonance energy transfer (FRET) analysis to fully unfolded staphylococcal nuclease. Artificial amino acids labeled with a FRET donor or acceptor were introduced by an amber codon and a four-base codon respectively. Eight double-labeled proteins were prepared, purified, and subjected to FRET analysis in 6 M urea. The observed behavior could be explained by a power law, R = αN0.44, where R, and N are the distance and the number of residues between donor and acceptor, and α is a coefficient. The index was smaller than the value expected for an excluded-volume random coil, 0.588, indicating that the fully unfolded proteins were more compact than polypeptides in good solvent. The FRET efficiency in the native state did not necessarily correlate to the distance obtained from crystal structure, suggesting that other factors such as the orientation factor made a substantial contribution to FRET.

Lectins are important not only as biological functional molecules but as useful probes for glycan analysis. Here, we developed fluorescent-labeled lectins showing fluorescence changes upon ligand-b...

Nonnatural amino acid mutagenesis using expanded genetic codes allows us to introduce artificial functional groups such as fluorophores at specific sites of proteins. By applying this technique, site-specific fluorescence labeling has been achieved. This method enables to generate fluorescent protein probes that show ligand-dependent fluorescence change based on fluorescence quenching of the incorporated fluorophores by endogenous Trp residues. This strategy has been proved to be effective using ligand-binding proteins and antibodies. Moreover, site-specific double-labeling of ligand-binding proteins with two fluorophores using two expanded genetic codons has achieved ratiometric detection of the ligand binding based on FRET and fluorescence quenching. These achievements demonstrate the usefulness of the site-specific incorporation of fluorescent nonnatural amino acids and their potential utility as research and diagnostic tools.

Novel light-emitting protein probes were designed and synthesized by incorporating fluorescent nonnatural amino acids and fusing light-emitting proteins. Gaussia luciferase was fused as a BRET dono...

Fluorescent indicators for protein phosphorylation are very important in not only fundamental biology but also biomedical applications. In this study, we developed novel fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine (pTyr) derivatives. A single-chain antibody variable fragment (scFv) against phosphotyrosine was fluorescent-labeled by incorporation of tetramethylrhodamine (TAMRA)-linked nonnatural amino acid at the N- or C-terminus. The TAMRA-labeled scFv showed fluorescence enhancement upon addition of pTyr-containing peptides based on antigen-dependent fluorescence quenching effect on TAMRA. The TAMRA-labeled scFv was further fused with enhanced green fluorescent protein (EGFP) to generate a double-labeled scFv for pTyr. In the absence of antigen, fluorescence resonance energy transfer (FRET) occurred from EGFP to TAMRA but TAMRA was quenched. The antigen-binding removed the quenching of TAMRA while FRET occurred without altering its efficiency. As a result of the FRET and antigen-dependent fluorescence quenching effect, the double-labeled scFv exhibited fluorescence ratio enhancement upon the antigen-binding. The fluorescent and fluorescent ratiometric indicators obtained in this study will become a novel tool for analysis of protein phosphorylation. Moreover, this strategy utilizes antibody derivatives, and therefore, can be easily applied to other antigen-antibody pairs to generate fluorescent ratiometric indicators for various target molecules.

The preparation of posttranslationally modified proteins is required to investigate the function and structure of modified proteins. However, homogeneously modified proteins are not easily isolated from natural sources or prepared using modification enzymes. Non-natural amino acid mutagenesis has enabled us to incorporate modified amino acids into specific positions of proteins in both cell-free and in-cell translation systems using tRNAs that are aminoacylated with modified amino acids. Here, we developed a method of double incorporation of modified amino acids and fluorescent non-natural amino acids in a quantitative, position-specific manner to obtain modified and fluorescently labeled proteins. To introduce methyllysine, dimethyllysine, trimethyllysine, and acetyllysine, frameshift and amber suppressor tRNAs aminoacylated with modified lysines were synthesized by chemical aminoacylation and supplied to an Escherichia coli cell-free translation system. The immunodetection of the translation products indicated that the modified lysines were incorporated into streptavidin and histone H3 in a quantitative, position-specific manner. Calmodulin derivatives containing a fluorescent non-natural amino acid at the N-terminal region and modified lysines at the Lys115 position were also synthesized, and their binding activity to a calmodulin-binding peptide was analyzed by fluorescence correlation spectroscopy. The results obtained here demonstrate that this method is useful in preparing and analyzing naturally occurring and non-natural modified proteins.

Fluorescence labeling is a useful technique for structural and functional analyses of proteins. In a previous study, we developed position-specific incorporation of visible wavelength fluorescent non-natural amino acids carrying relatively small BODIPY fluorophores into proteins, in response to a four-base codon CGGG. Here, we have expanded this position-specific fluorescence labeling method to include relatively large non-natural amino acids carrying photostable rhodamine dyes. TAMRA-linked aminophenylalanine was synthesized and attached to a tRNA having a four-base anticodon, and its incorporation into proteins was examined in an Escherichia coli cell-free translation system. TAMRA-labeled amino acids were successfully incorporated into proteins, although incorporation was allowed only at the N-terminal region. Insertion of two codons encoding a stop codon in the + 1 frame before four-base codon suppressed the expression of non-labeled proteins that may have been produced by spontaneous + 1 frameshift upstream of the four-base codon. Alternation of the incorporation position affected the expression level of the TAMRA-labeled protein. In addition, alternation of upstream and downstream codons affected the efficiency and accuracy of TAMRA-labeled amino acid incorporation. Based on these results, a novel tag peptide was developed; it contained the four-base codon at the 9th position with optimized upstream and downstream codons. This tag peptide was effective for producing proteins with various fluorescent labels at the N-terminal region.

Incorporation of non-natural amino acid derivatives containing fluorescent groups into proteins is a useful method for protein analyses. Here, we investigated the incorporation of fluorescent-label...

Incorporation of non-natural amino acids into proteins is a useful method for protein researches, including position-specific labeling with fluorophores and biotin. Here, we incorporated single non...

Incorporation of non-natural amino acids into proteins in response to amber or four-base codons is a useful technology for protein research. In the case of the amber codon, however, release factor 1 can competitively decode the same codon, and consequently inhibit the incorporation of non-natural amino acids. To improve amber codon-mediated incorporation, we carried out a comprehensive screening of amber suppressor tRNAs derived from all tRNAs encoded in the genomes of Escherichia coli K12 and Mycoplasma capricolum. The amber suppressor tRNAs were synthesized from synthetic genes, aminoacylated with a fluorescent non-natural amino acid, and added to an E. coli cell-free translation system. Fluorescent SDS-PAGE analysis indicated that Trp tRNAs showed high suppressor activity in both organisms. Further mutagenesis and screening revealed that M. capricolum Trp(1) tRNA with G1C72A73 mutation is the most suitable for efficient and specific incorporation of non-natural amino acids into proteins in response to the amber codon.

We have developed a novel method to attach a fluorescent label at the N terminus of proteins through a four-base codon-mediated incorporation of a fluorescent hydroxy acid and subsequent cleavage of the ester bond in a cell-free translation system. We found that a fluorescent-labeled p-amino-L-phenyllactic acid was successfully incorporated downstream of N-terminal tag peptides in response to a CGGG codon, and the tag peptides could be removed through ester cleavage to leave the fluorescent hydroxy acid at the N terminus of the proteins. Immunoprecipitation analysis revealed that ester cleavage occurred spontaneously during the translation reaction. The efficiency of the ester cleavage and the yield of the labeled proteins were dependent on the peptide tag sequence. We demonstrate that the insertion of an asparagine residue between the N-terminal T7 tag and the fluorescent hydroxy acid achieved both quantitative ester cleavage and efficient expression of the labeled proteins. The present method is a potential tool for N-terminal specific labeling of proteins with various compounds.

Saturation mutagenesis is a useful technique for the structural and functional analyses of proteins and for protein engineering. However, the extensive mutagenesis of genes and expression of mutated proteins are tedious and time-consuming. We have developed a simple and rapid method for the expression of mutated proteins with comprehensive single amino acid substitutions from single mutated genes having a four-base codon in a cell-free translation system. Twenty types of tRNA that were aminoacylated with one of the 20 proteinogenic amino acids and that contained a four-base anticodon were prepared by chemical aminoacylation. In the presence of one of the aminoacyl-tRNAs, a streptavidin mRNA with a four-base codon at the Tyr83 position was expressed in an Escherichia coli cell-free translation system. The N-terminus of the expressed proteins was fluorescently labeled using a fluorescent-labeled initiator Met-tRNA. Fluorescence imaging of an SDS-PAGE gel showed that all the amino acids are incorporated in response to the four-base codon; however, the incorporation efficiency was dependent on the structure of the side chains. Streptavidin mutants with comprehensive amino acid substitutions at the Tyr83, Arg84, and Tyr54 positions were used for analyzing their biotin-binding activity by dot blot analysis. These results demonstrate that this method is effective for the expression and analysis of mutated proteins with comprehensive amino acid substitutions at desired positions.

Biotinylation is useful for the detection, purification and immobilization of proteins. It is performed by chemical modification, although position-specific and quantitative biotinylation is rarely achieved. We developed a position-specific biotinylation method using biotinylated non-natural amino acids. We showed that biotinylated p-aminophenylalanine derivatives were incorporated into a protein more efficiently than biotinylated lysine derivatives in a cell-free translation system. In addition, the biotinylated p-aminophenylalanines overcame the serious position-dependency observed for biotinylated lysines. The present method will be useful for detection and purification of proteins along with comprehensive exploration of surface-exposed residues and oriented immobilization of proteins.

In the present study, a novel molecular biosensor system model was designed by using a couple of the fluorescent unnatural mutant streptavidin and the carbazole-labeled biotin. BODIPY-FL-aminophenylalanine (BFLAF), a fluorescent unnatural amino acid was position-specifically incorporated into Trp120 position of streptavidin by four-base codon method. On the other hand, carbazole-labeled biotin was synthesized as a quencher for the fluorescent Trp120BFLAF mutant streptavidin. The fluorescence of fluorescent Trp120BFLAF mutant streptavidin was decreased as we expected when carbazole-labeled biotin was added into the mutant streptavidin solution. Furthermore, the fluorescence decrease of Trp120BFLAF mutant streptavidin with carbazole-labeled biotin (100 nM) was recovered by the competitive addition of natural biotin. This result demonstrated that by measuring the fluorescence quenching and recovery, a couple of the fluorescent Trp120BFLAF mutant streptavidin and the carbazole-labeled biotin were successfully applicable for quantification of free biotin as a molecular biosensor system. Copyright © 2016 John WileySons, Ltd.

Methanosarcina species pyrrolysyl-tRNA synthetase (PylRS) attaches Pyl to its cognate amber suppressor tRNA. The introduction of two mutations (Y384F and Y306A) into PylRS was previously shown to generate a mutant, designated LysZ-RS, that was able to attach N-benzyloxycarbonyl-L-lysine (LysZ) to its cognate tRNA. Despite the potential of LysZ derivatives, further LysZ-RS engineering has not been performed; consequently, we aimed to generate LysZ-RS mutants with improved LysZ incorporation activity through in vitro directed evolution. Using a liposome-based in vitro compartmentalization (IVC) approach, we screened a randomly mutagenized gene library of LysZ-RS and obtained a mutant that showed increased LysZ incorporation activity both in vitro and in vivo. The ease and high flexibility of liposome-based IVC should enable the evolution of not only LysZ-RS that can attach various LysZ derivatives but also of other enzymes involved in protein translation.

We designed and synthesized new, fluorescent, non-natural amino acids that emit fluorescence of wavelengths longer than 500 nm and are accepted by an Escherichia coli cell-free translation system. We synthesized p-aminophenylalanine derivatives linked with BODIPY fluorophores at the p-amino group and introduced them into streptavidin using the four-base codon CGGG in a cell-free translation system. Practically, the incorporation efficiency was high enough for BODIPYFL, BODIPY558 and BODIPY576. Next, we incorporated BODIPYFL-aminophenylalanine and BODIPY558-aminophenylalanine into different positions of calmodulin as a donor and acceptor pair for fluorescence resonance energy transfer (FRET) using two four-base codons. Fluorescence spectra and polarization measurements revealed that substantial FRET changes upon the binding of calmodulin-binding peptide occurred for the double-labeled calmodulins containing BODIPY558 at the N terminus and BODIPYFL at the Gly40, Phe99 and Leu112 positions. These results demonstrate the usefulness of FRET based on the position-specific double incorporation of fluorescent amino acids for analyzing conformational changes of proteins.

The general and efficient method for the site-directed glycosylation of proteins is a key step in order to understand the biological importance of the carbohydrate chains of proteins and to control functional roles of the engineered glycoproteins in terms of the development of improved glycoprotein therapeutics. We have developed a novel method for site-directed glycosylation of proteins based on chemoselective blotting of common reducing sugars by genetically encoded proteins. The oxylamino-functionalized L-homoserine residues, 2-amino-4-O-(N-methylaminooxy) butanoic acid and 2-amino-4-aminooxy butanoic acid, were efficiently incorporated into proteins by using the four-base codon/anticodon pair strategy in Escherichia coli in vitro translation. Direct and chemoselective coupling between unmodified simple sugars and N-methylaminooxy group displayed on the engineered streptavidin allowed for the combinatorial synthesis of novel glycoprotein mimetics.

Techniques for position-specific incorporation of non-natural amino acids in an in vitro protein synthesizing system are described. First, a PNA-assisted non-enzymatic tRNA aminoacylation with a variety of natural and non-natural amino acids is described. With this technique, one can aminoacylate a specific tRNA simply by adding a preformed amino acid activated ester-PNA conjugate into an in vitro protein biosynthesizing system. Second, the genetic code is expanded by introducing 4-base codons that can be exclusively translated to non-natural amino acids. The most advantageous point of the 4-base codon strategy is to introduce multiple amino acids into specific positions in single proteins by using mutually orthogonal 4-base codons and orthogonal tRNAs. An easy and quick method for preparation of tRNAs possessing 4-base anticodons is also described. Combination of the non-enzymatic aminoacylation and the 4-base codon/anticodon strategy gives an easy and widely applicable technique for incorporating a variety of non-natural amino acids into proteins in vitro.

Incorporation of nonnatural amino acids into specific positions of proteins was achieved by using extended codons such as CGGG four-base codon. Aminoacyl-tRNAs having nonnatural amino acids and fou...

Factors that affect the efficiency of in vitro synthesis of mutant proteins that contain nonnatural amino acids were investigated. The process of the nonnatural mutagenesis consists of chemical aminoacylation of a tRNA that contains a 4-base anticodon, followed by in vitro synthesis in the presence of an mRNA that contains the corresponding 4-base codon. Detailed studies on the time courses of the synthesis revealed two major factors that suppress the yield of nonnatural mutants compared with the wild-type protein. First, a cyclic tRNA that exists as a by-product of the chemical aminoacylation inhibits the protein synthesis. Second, the very short lifetime of a tRNA aminoacylated with a nonnatural amino acid limits the protein yield. As a simple and practical way of surmounting these factors, aminoacyl tRNA was added into the in vitro system at 5 min after the start of the synthesis. The addition increased the protein yield up to the level of conventional proteins in the in vitro system.

Chemical and biological diversity of protein structures and functions can be widely expanded by position-specific incorporation of non-natural amino acids carrying a variety of specialty side groups. After the pioneering works of Schultz's group and Chamberlin's group in 1989, noticeable progress has been made in expanding types of amino acids, in finding novel methods of tRNA aminoacylation and in extending genetic codes for directing the positions. Aminoacylation of tRNA with non-natural amino acids has been achieved by directed evolution of aminoacyl-tRNA synthetases or some ribozymes. Codons have been extended to include four-base codons or non-natural base pairs. Multiple incorporation of different non-natural amino acids has been achieved by the use of a different four-base codon for each tRNA. The combination of these novel techniques has opened the possibility of synthesising non-natural mutant proteins in living cells.

Four-base codon strategy was applied to incorporate a fluorophore-quencher pair into specific positions on a single protein; beta-anthraniloyl-L-alpha,beta-diaminopropionic acid (atnDap) was employed as a fluorophore and p-nitrophenylalanine (ntrPhe) as a quencher. Their positions were directed by the CGGG/CCCG and GGGC/CCCG four-base codon/anticodon pairs and two doubly mutated streptavidins, i.e., ((52)atnDap, (84)ntrPhe) and ((54)ntrPhe, (84)atnDap) mutants were synthesized through Escherichia coli in vitro protein synthesizing systems. Intramolecular photoinduced electron transfer (ET) was observed as the decrease of intensity in steady-state fluorescence spectroscopy and as the shortening of fluorescence decaytimes. The quenching data indicated that the ET rate reflects the detailed structure of the protein.

General preparation of glycosylated amino acylated nucleotide for in vitro peptide synthesis was described. Both O-glycosylated amino acyl nucleotides and C-glycosylated amino acyl nucleotide were synthesized by choosing the appropriate protecting group.

A general method was developed for the construction of a library of mutant genes. The method, termed random insertion/deletion (RID) mutagenesis, enables deletion of an arbitrary number of consecutive bases at random positions and, at the same time, insertion of a specific sequence or random sequences of an arbitrary number into the same position. The applicability of the RID mutagenesis was demonstrated by replacing three randomly selected consecutive bases by the BglII recognition sequence (AGATCT) in the GFPUV gene. In addition, the randomly selected three bases were replaced by a mixture of 20 codons. These mutants were expressed in Escherichia coli, and those that showed fluorescence properties different from the wild-type GFP were selected. A yellow fluorescent protein and an enhanced green fluorescent protein, neither of which could be obtained by error-prone PCR mutagenesis, were found among the six mutants selected. Several mutants of the DsRed protein that show different fluorescence properties were also obtained.

Position-specific incorporation of nonnatural amino acids into proteins (nonnatural mutagenesis) via an in vitro protein synthesizing system was applied to incorporate a variety of amino acids carrying specialty side groups. A list of nonnatural amino acids thus far successfully incorporated through in vitro translation systems is presented. The position of nonnatural amino acid incorporation was directed by four-base codon/anticodon pairs such as CGGG/CCCG and AGGU/ACCU. The four-base codon strategy was more efficient than the amber codon strategy and could incorporate multiple nonnatural amino acids into single proteins. This multiple mutagenesis will find wide applications, especially in building paths of electron transfer on proteins. The extension of translation systems by the introduction of nonnatural amino acids, four-base codon/anticodon pairs, orthogonal tRNAs, and artificial aminoacyl tRNA synthetases, is a promising approach towards the creation of "synthetic microorganisms" with specialty functions.

Fluorescent nonnatural amino acids were incorporated into specific positions of streptavidin. The positions of the nonnatural amino acids were directed by a CGGG/CCCG four-base codon/anticodon pair. The nonnatural mutants with a single 2-anthrylalanine at the 22nd, 43rd, 54th, and 120th positions, respectively, were found to bind biotin, indicating that the mutants retained active conformation. The fluorescence intensities of the anthryl groups were relatively insensitive to the positions and the biotin binding when excited at 265 nm. When the anthryl group at the 120th position was excited through energy transfer from tryptophan units, the fluorescence intensity markedly decreased with biotin binding, because of a suppression of the energy transfer. Amino acids carrying 7-methoxycoumarine fluorophore were also incorporated at the 120th position. Their fluorescence quantum yields were very sensitive to the biotin binding. The high sensitivity of the coumarine-labeled streptavidin exemplifies potential applications of fluorescent nonnatural mutants for detecting specific molecules at very low concentrations.

Site-directed incorporation of PEGylated nonnatural amino acids with 4, 8, and 12 repeated ethylene glycol units was examined in a cell-free translation system. PEGylated aminophenylalanine derivatives were successfully incorporated into proteins, whereas PEGylated lysines were not. The incorporation efficiency of the PEGylated amino acids decreased with an increase in PEG chain length. The present method will be useful for preparation of proteins which are PEGylated in a site-specific and quantitative manner.

Site-specific mutagenesis of proteins was extended to incorporate nonnatural amino acids through in vitro cell-free translation or through in vivo translation in the living cells. To this end, a va...

Efficiencies of the incorporation of various nonnatural amino acids carrying aromatic side groups into streptavidin were examined. The aromatic amino acids were linked to a mixed dinucleotide, pdCpA, and the resulting aminoacyl pdCpAs were coupled with tRNAcccg(−CA) to afford chemically aminoacylated tRNAcccg's. Mutant streptavidin mRNA containing a CGGG 4 base codon at the Tyr83 site was prepared and added to an Escherichia coli in vitro translation system with the aminoacyl tRNAcccg. The expression of the full-length mutant streptavidins was confirmed by a Western blot analysis, and their biotin binding activity was examined by a dot blot analysis. The Western blot analysis indicated that the efficiencies of the incorporation were higher for aromatic groups with straight configurations than those with widely expanded or bend configurations. The incorporation efficiencies were also examined in a rabbit reticulocyte lysate. In the latter system, the efficiencies were markedly improved for nonnatural amino...

Site-directed mutagenesis of streptavidin was carried out using a frame-shift suppressor tRNA that is aminoacylated with l-p-nitrophenylalanine and has a CCCG 4-base anticodon. Streptavidins carrying a single p-nitrophenylalanine at 22 different sites were prepared from mRNAs that contain a single CGGG 4-base codon at the mutation site. Of the 22 mutants, 14 mutants were found to bind N-biotinyl-l-1-pyrenylalanine. Site-to-site photoinduced electron transfer from the excited pyrenyl group to the nitrophenyl group was observed with steady-state fluorescence spectroscopy as well as by fluorescence decay measurement. The rate constants of the electron transfer decreased with the edge-to-edge distances that were predicted from the X-ray crystallographic structure of streptavidin. The distance dependence was analyzed on the basis of the tunneling pathway model. It was found that the rate constants are compatible with those in other proteins carrying metal complexes, except for the cases where electron transfer...

O-GalNAcα-modified proteins are the precursor of mucin-type O-glycosylated proteins. Homogeneously O-glycosylated proteins are required to investigate the biological functions of glycoproteins and to develop biopharmaceuticals. Here we show that the incorporation of GalNAcα-Thr into proteins successfully proceeded by the use of a chemically aminoacylated tRNA. GalNAcα-Thr was chemoenzymatically attached to amber suppressor tRNA and the product was subjected to in vitro translation together with streptavidin mRNA containing the UAG codon. Gel electrophoresis and mass analysis showed that GalNAcα-Thr was successfully incorporated into the N-terminus, although it was not incorporated at the interior. This method will facilitate the preparation of homogeneous GalNAcα-proteins.

Saturation mutagenesis of proteins, in which an amino acid at a specific site is substituted with each of the other 19 amino acids, is a powerful method for protein analysis and engineering. However, 19 mutated genes have to be prepared to express all possible amino acid-substituted proteins at one site. We previously reported a four-base codon-mediated saturation mutagenesis method for the expression of all 20 amino acid-substituted proteins from one four-base codon-containing gene using 20 types of chemically aminoacylated tRNAs corresponding to the four-base codon. In this study, an improved method for saturation mutagenesis using an amber codon was developed. By combining the use of Escherichia coli-derived amber suppressor tRNAs and chemically aminoacylated Mycoplasma-derived tRNAs, all 20 mutated proteins were successfully expressed from one amber mutant gene in a cell-free translation system. The use of E. coli-derived amber suppressor tRNAs simplified the preparation of the tRNA reagents required for saturation mutagenesis, and also improved the expression of some of the mutated proteins. The expressed mutant proteins were used to evaluate the effect of the amino acid substitutions on the ligand-binding activity. To further expand the possibilities of saturation mutagenesis, a series of nonnatural amino acids analogous to a naturally occurring amino acid was added to the amino acid repertoire. The expanded saturation mutagenesis was utilized to evaluate the effect of a series of atomic-level side chain substitutions on the protein activity.

Unnatural amino acid mutagenesis allows us to introduce unnatural α-amino acids into internal positions of proteins in response to expanded codons such as amber and four-base codons. To improve the unnatural amino acid mutagenesis, the incorporation of unnatural α-amino acids and non-α-amino acids into the N-terminus of proteins has been achieved using expanded initiation codons. Here, we describe the method for the incorporation of fluorescent-labeled non-α-amino acids into the N-terminus of proteins in a cell-free translation system.