Publications by authors named "Kensaku Sakamoto"

Antibodies are widely used as therapeutic agents to tackle various diseases. In the present study, to enhance their clinical values, we rationally designed pH-responsivity by exploiting the idiosyncratic protonation/deprotonation profiles of non-natural amino acids. 3-Nitro-L-tyrosine, 3-cyano-L-tyrosine, and 3, 5-halogenated-L-tyrosine, each with near neutral pK, were thus incorporated into Fab fragments in place of tyrosines and other residues in the variable regions.

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Identification of protein-protein interfaces is necessary for understanding and regulating biological events. Genetic code expansion enables site-specific photo-cross-linking by introducing photo-reactive non-canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein-protein interactions and is applicable in mammalian cells.

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Cell-free molecular display techniques have been utilized to select various affinity peptides from peptide libraries. However, conventional techniques have difficulties associated with the translational termination through in-frame UAG stop codons and the amplification of non-specific peptides, which hinders the desirable selection of low-affinity peptides. To overcome these problems, we established a scheme for ribosome display selection of peptide epitopes bound to monoclonal antibodies and then applied genetic code expansion with synthetic X-tRNA reprogramming of the UAG codons (X = Tyr, Trp, or -benzoyl-l-phenylalanine (Bzo-Phe)) to the scheme.

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Genetic code expansion enables site-specific photo-crosslinking by introducing photo-reactive non-canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein-protein interactions and is applicable in mammalian cells. However, the identification of the crosslinked region still remains challenging.

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Pairs of pyrrolysyl-tRNA synthetase (PylRS) and tRNA from and are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). Previously, we achieved full productivity of cell-free protein synthesis for bulky non-canonical amino acids, including -(((()-cyclooct-2-en-1-yl)oxy)carbonyl)-L-lysine (TCO*Lys), by using PylRS with structure-based mutations in and around the amino acid binding pocket (first-layer and second-layer mutations, respectively). Recently, the PylRS·tRNA pair from a methanogenic archaeon ISO4-G1 was used for genetic code expansion.

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This Special Issue is intended to highlight recent advances in genetic code expansion, particularly the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins [...

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Chaperone-mediated autophagy (CMA) is a unique proteolytic pathway, in which cytoplasmic proteins recognized by heat shock cognate protein 70 (Hsc70/HSPA8) are transported into lysosomes for degradation. The substrate/chaperone complex binds to the cytosolic tail of the lysosomal-associated membrane protein type 2A (LAMP2A), but whether the interaction between Hsc70 and LAMP2A is direct or mediated by other molecules has remained to be elucidated. The structure of LAMP2A comprises a large lumenal domain composed of two domains, both with the β-prism fold, a transmembrane domain and a short cytoplasmic tail.

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Protein engineering and design principles employing the 20 standard amino acids have been extensively used to achieve stable protein scaffolds and deliver their specific activities. Although this confers some advantages, it often restricts the sequence, chemical space, and ultimately the functional diversity of proteins. Moreover, although site-specific incorporation of non-natural amino acids (nnAAs) has been proven to be a valuable strategy in protein engineering and therapeutics development, its utility in the affinity-maturation of nanobodies is not fully explored.

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Most of the currently approved therapeutic antibodies are of the immunoglobulin gamma (IgG) κ isotype, leaving a vast opportunity for the use of IgGλ in medical treatments. The incorporation of designer amino acids into antibodies enables efficient and precise manufacturing of antibody chemical conjugates. Useful conjugation sites have been explored in the constant domain of the human κ-light chain (LCκ), which is no more than 38% identical to its LCλ counterpart in amino acid sequence.

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In this study, we developed a simulation code powered by lattice dose-response functions (hereinafter SIBYL), which helps in the quick and accurate estimation of external gamma-ray doses emitted from a radioactive plume and contaminated ground. SIBYL couples with atmospheric dispersion models and calculates gamma-ray dose distributions inside a target area based on a map of activity concentrations using pre-evaluated dose-response functions. Moreover, SIBYL considers radiation shielding due to obstructions such as buildings.

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Expansion of the amino-acid repertoire with synthetic derivatives introduces novel structures and functionalities into proteins. In this study, we improved the antigen binding of antibodies by incorporating halogenated tyrosines at multiple selective sites. Tyrosines in the Fab fragment of an anti-EGF-receptor antibody 059-152 were systematically replaced with 3-bromo- and 3-chlorotyrosines, and simultaneous replacements at four specific sites were found to cause a tenfold increase in the affinity toward the antigen.

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Pyrrolysyl-tRNA synthetase (PylRS)/tRNA pairs from and are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). In this study, we achieved the full productivity of cell-free protein synthesis for difficult, bulky non-canonical amino acids, such as -(((()-cyclooct-2-en-1-yl)oxy)carbonyl)-l-lysine (TCO*Lys), by using PylRS. First, based on the crystal structure of PylRS, the productivities for various non-canonical amino acids were greatly increased by rational engineering of the amino acid-binding pocket.

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Pyrrolysyl-tRNA synthetase (PylRS) and tRNA have been extensively used for genetic-code expansion. A Methanosarcina mazei PylRS mutant bearing the Y306A and Y384F mutations (PylRS(Y306A/Y384F)) encodes various bulky non-natural lysine derivatives by UAG. In this study, we examined how PylRS(Y306A/Y384F) recognizes many amino acids.

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Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the strain RFzero-iy, which is engineered to lack release factor 1 (RF-1).

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The L-shape form of tRNA is maintained by tertiary interactions occurring in the core. Base changes in this domain can cause structural defects and impair tRNA activity. Here, we report on a method to safely engineer structural variations in this domain utilizing the noncanonical scaffold of tRNA.

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In the present study, we attempted to control the pH profile of the catalytic activity of the industrially relevant alkaline protease KP-43, by incorporating 3-nitro-l-tyrosine and 3-chloro-l-tyrosine at and near the catalytic site. Thirty KP-43 variants containing these non-natural amino acids at the specific positions were synthesized in host cells with expanded genetic codes. The variant with 3-nitrotyrosine at position 205, near the substrate binding site, retained its catalytic activity at the neutral pH and showed a 60% activity reduction at pH 10.

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Genetic code expansion has largely relied on two types of the tRNA-aminoacyl-tRNA synthetase pairs. One involves pyrrolysyl-tRNA synthetase (PylRS), which is used to incorporate various lysine derivatives into proteins. The widely used PylRS from Methanosarcinaceae comprises two distinct domains while the bacterial molecules consist of two separate polypeptides.

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Escherichia coli β-lactamase TEM-1 is potentially useful in the antibody-directed enzyme/prodrug therapy (ADEPT), converting nontoxic prodrugs to toxic agents. The produced toxin would kill cancer cells, when the enzyme is attached to a tumor-antigen-specific antibody. However, the off-site reaction possibly occurring in the blood or normal tissues raises safety concern.

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The bromodomain and extra-terminal domain (BET) proteins are promising drug targets for cancer and immune diseases. However, BET inhibition effects have been studied more in the context of bromodomain-containing protein 4 (BRD4) than BRD2, and the BET protein association to histone H4-hyperacetylated chromatin is not understood at the genome-wide level. Here, we report transcription start site (TSS)-resolution integrative analyses of ChIP-seq and transcriptome profiles in human non-small cell lung cancer (NSCLC) cell line H23.

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In the present study, we simultaneously incorporated two types of synthetic components into microbial transglutaminase (MTG) from Streptoverticillium mobaraense to enhance the utility of this industrial enzyme. The first amino acid, 3-chloro-l-tyrosine, was incorporated into MTG in response to in-frame UAG codons to substitute for the 15 tyrosine residues separately. The two substitutions at positions 20 and 62 were found to each increase thermostability of the enzyme, while the seven substitutions at positions 24, 34, 75, 146, 171, 217, and 310 exhibited neutral effects.

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The genetic code in bacteria and animal cells has been expanded to incorporate novel amino acids into proteins. Recent efforts have enabled genetic code expansion in nematodes, flies, and mice, whereas such engineering is rare with industrially useful animals. In the present study, we engineered the silkworm Bombyx mori to synthesize silk fiber functionalized with azidophenylalanine.

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Cell-free protein synthesis is a useful method for preparing proteins for functional or structural analyses. However, batch-to-batch variability with regard to protein synthesis activity remains a problem for large-scale production of cell extract in the laboratory. To address this issue, we have developed a novel procedure for large-scale preparation of bacterial cell extract with high protein synthesis activity.

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The site-specific chemical conjugation of proteins, following synthesis with an expanded genetic code, promises to advance antibody-based technologies, including antibody drug conjugation and the creation of bispecific Fab dimers. The incorporation of non-natural amino acids into antibodies not only guarantees site specificity but also allows the use of bio-orthogonal chemistry. However, the efficiency of amino acid incorporation fluctuates significantly among different sites, thereby hampering the identification of useful conjugation sites.

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