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Biomedical subjects

Takahiro Hohsaka

Publications and source records attributed to Takahiro Hohsaka.

At least 19 recordsLinked to original sources
PubMed2006

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.

Amino Acids↗
PubMed2006

Four-base codon mediated mRNA display to construct peptide libraries that contain multiple nonnatural amino acids.

In vitro selection and directed evolution of peptides from mRNA display are powerful strategies to find novel peptide ligands that bind to target biomolecules. In this study, we expanded the mRNA display method to include multiple nonnatural amino acids by introducing three different four-base codons at a randomly selected single position on the mRNA. Another nonnatural amino acid may be introduced by suppressing an amber codon that may appear from a (NNK)(n) nucleotide sequence on the mRNA. The mRNA display was expressed in an Escherichia coli in vitro translation system in the presence of three types of tRNAs carrying different four-base anticodons and a tRNA carrying an amber anticodon, the tRNAs being chemically aminoacylated with different nonnatural amino acids. The complexity of the starting mRNA-displayed peptide library was estimated to be 1.1 x 10(12) molecules. The effectiveness of the four-base codon mediated mRNA display method was demonstrated in the selection of biocytin-containing peptides on streptavidin-coated beads. Moreover, a novel streptavidin-binding nonnatural peptide containing benzoylphenylalanine was obtained from the nonnatural peptide library. The nonnatural peptide library from the four-base codon mediated mRNA display provides much wider functional and structural diversity than conventional peptide libraries that are constituted from 20 naturally occurring amino acids.

Amino Acids↗
PubMed2006

FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids.

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.

Calmodulin↗
PubMed2006

Development of amber suppressor tRNAs appropriate for incorporation of nonnatural amino acids.

An amber suppression method has been used for incorporation of nonnatural amino acids into proteins. However, the incorporation efficiency of nonnatural amino acids through an amber codon has been low, which restricts the application of the proteins containing nonnatural amino acids. In this study, we screened a wide variety of amber suppressor tRNAs to discover tRNAs capable to incorporate nonnatural amino acids with high efficiency. To this purpose, synthetic amber suppressor tRNAs of E. coli and Mycoplasma capricolum were screened for the incorporation of a fluorescently labeled nonnatural amino acid in an E. coli cell-free translation system. tRNAs that showed high capability for the incorporation were then mutated not to be aminoacylated by any of endogenous aminoacyl-tRNA synthetases of E. coli and to enhance the incorporation capability. As a result of these investigations, we successfully obtained several amber suppressor tRNAs with high ability for the incorporation of nonnatural amino acids.

Amino Acids↗
PubMed2005

Molecular design of glycoprotein mimetics: glycoblotting by engineered proteins with an oxylamino-functionalized amino acid residue.

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.

Amino Acids↗
PubMed2005

A small-molecule-based approach to sense codon-templated natural-unnatural hybrid peptides. Selective silencing and reassignment of the sense codon by orthogonal reacylation stalling at the single-codon level.

In the presence of the stable sulfamoyl analogue of phenylalanyl adenylate (Phe-SA), the UUU/UUC sense codon for phenylalanine (Phe) can be silenced and reassigned to a naphthylalanine (Nap) conjugated to tRNAPhe. We have demonstrated the efficiency and selectivity or orthogonality of the Phe-to-Nap reassignment induced by an "orthogonal reacylation stalling" strategy at the single-codon level in the translation of mRNAs of dihydrofolate reductase and a 24-mer oligopeptide. We used a prokaryotic translation system with an essential preincubation, during which the endogenous precharged phenylalanyl-tRNAPhe undergoes deacylation and the reacylation of the resulting tRNAPhe is stalled by the action of Phe-SA to inhibit the phenylalanyl-tRNA synthetase activity. We discuss the significance of the present small-molecule-based approach to sense-codon templated natural-unnatural peptides.

Acylation↗
PubMed2005

Synthesis and sequence optimization of GFP mutants containing aromatic non-natural amino acids at the Tyr66 position.

In order to alter the fluorescence properties of green fluorescent protein (GFP), aromatic non-natural amino acids were introduced into the Tyr66 position of GFP in a cell-free translation system using a four-base codon method. Two non-natural mutants (O-methyltyrosine and p-aminophenylalanine mutants) out of 18 mutants showed blue-shifted but weak fluorescence compared with wild-type GFP. Then the aminophenylalanine mutant was sequence optimized by introducing random mutations around the Tyr66 site. For this purpose, a method for random mutation of non-natural proteins in a cell-free system was developed. Three aminophenylalanine mutants with Y145F, Y145L and Y145 M mutations were obtained, which exhibited increased fluorescence by 1.5-, 3- and 4-fold, respectively. These results indicate that random mutation around non-natural amino acids is useful strategy in order to improve protein functions that are reduced by non-natural amino acid incorporation. The method described here will be applicable to other non-natural mutant proteins in a high-throughput manner.

Cell-Free System↗
PubMed2005

Four-base codon/anticodon strategy and non-enzymatic aminoacylation for protein engineering with non-natural amino acids.

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.

Amino Acids↗
PubMed2005

Four-base codon-mediated incorporation of non-natural amino acids into proteins in a eukaryotic cell-free translation system.

Various four-base codons have been shown to work for the introduction of non-natural amino acids into proteins in an Escherichia coli cell-free translation system. Here, a four-base codon-mediated non-natural mutagenesis was applied to a eukaryotic rabbit reticulocyte cell-free translation system. Mutated streptavidin mRNAs containing four-base codons were prepared and added to a rabbit reticulocyte lysate in the presence of tRNAs that were aminoacylated with a non-natural amino acid and had the corresponding four-base anticodons. A Western blot analysis of translation products indicated that the four-base codons CGGU, CGCU, CCCU, CUCU, CUAU, and GGGU were efficiently decoded by the aminoacyl-tRNAs having the corresponding four-base anticodons. In contrast, the four-base codons AGGU, AGAU, CGAU, UUGU, UCGU, and ACGU were not decoded. The stop codon-derived four-base codons UAGU, UAAU, and UGAU were found to be inefficient, whereas the amber codon UAG and opal codon UGA were efficient for the incorporation of non-natural amino acids. The application of the expanded genetic code in a eukaryotic cell-free system opens the possibility of a four-base codon-mediated incorporation of non-natural amino acids into proteins in living eukaryotic cells.

Amino Acids↗
PubMed2004

Position-specific incorporation of dansylated non-natural amino acids into streptavidin by using a four-base codon.

Novel non-natural amino acids carrying a dansyl fluorescent group were designed, synthesized, and incorporated into various positions of streptavidin by using a CGGG four-base codon in an Escherichia coli in vitro translation system. 2,6-Dansyl-aminophenylalanine (2,6-dnsAF) was found to be incorporated into the protein more efficiently than 1,5-dansyl-lysine, 2,6-dansyl-lysine, and 1,5-dansyl-aminophenylalanine. Fluorescence measurements indicate that the position-specific incorporation of the 2,6-dnsAF is a useful technique to probe protein structures. These results also indicate that well-designed non-natural amino acids carrying relatively large side chains can be accepted as substrates of the translation system.

Amino Acids↗
PubMed2004

Efficient synthesis of nonnatural mutants in Escherichia coli S30 in vitro protein synthesizing system.

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.

Journal Article↗
PubMed2004

Site-directed incorporation of non-natural amino acid into Streptomyces xylanase.

A key residue, W26G existing around the substrate-binding cleft of beta-1,4-xylanase from Streptomyces olivaceoviridis E-86 (SoXyn10A), was ascertained in this study. Several kinds of W266 mutants substituted by Trp analogs were synthesized by a site-directed incorporation method to elucidate the functional role of this aromatic residue. Significant decreases of hydrolytic activities were observed in all mutants, even though the substitution of the aromatic side chain was not so drastic, suggesting that the W266 residue had a fatal role in the hydrolytic activity of SoXyn10A.

Amino Acids↗
PubMed2003

Incorporation of fluorescently labeled nonnatural amino acids into proteins in an E. coli in vitro translation system.

Various nonnatural amino acids has been incorporated into proteins by using four-base codons in an E. coli in vitro translation system. Here, design and synthesis of novel fluorescently labeled nonnatural amino acids and their incorporation into proteins were investigated. Transfer RNAs that contained a CCCG anticodon and were aminoacylated with BODIPY FL-labeled amino acids were prepared by a chemical aminoacylation method, and added to an in vitro translation system in the presence of a streptavidin mRNA containing a CGGG codon. SDS-PAGE and Western blot analysis of the synthesized proteins indicate that BODIPY FL-labeled aminophenylalanine derivatives are efficiently incorporated into proteins through the four-base codon decoding.

Amino Acids↗
PubMed2002

Position-specific incorporation of a fluorophore-quencher pair into a single streptavidin through orthogonal four-base codon/anticodon pairs.

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.

Amino Acids↗