Co-reporter:Atsushi Yamaguchi, Takayoshi Matsuda, Kazumasa Ohtake, Tatsuo Yanagisawa, Shigeyuki Yokoyama, Yoshihisa Fujiwara, Takayoshi Watanabe, Takahiro Hohsaka, and Kensaku Sakamoto
Bioconjugate Chemistry 2016 Volume 27(Issue 1) pp:198
Publication Date(Web):December 1, 2015
DOI:10.1021/acs.bioconjchem.5b00602
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 tRNAPyl. 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.
Co-reporter:Dr. Atsuko Uyeda;Dr. Takayoshi Watanabe;Dr. Yasuhiko Kato; Hajime Watanabe; Tetsuya Yomo; Takahiro Hohsaka;Dr. Tomoaki Matsuura
ChemBioChem 2015 Volume 16( Issue 12) pp:1797-1802
Publication Date(Web):
DOI:10.1002/cbic.201500174
Abstract
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.
Co-reporter:Naoki Shozen, Issei Iijima, Takahiro Hohsaka
Bioorganic & Medicinal Chemistry Letters 2009 Volume 19(Issue 17) pp:4909-4911
Publication Date(Web):1 September 2009
DOI:10.1016/j.bmcl.2009.07.105
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.
Co-reporter:Issei Iijima
ChemBioChem 2009 Volume 10( Issue 6) pp:999-1006
Publication Date(Web):
DOI:10.1002/cbic.200800703
Co-reporter:Takayoshi Watanabe;Yoichi Miyata;Ryoji Abe;Norihito Muranaka Dr.
ChemBioChem 2008 Volume 9( Issue 8) pp:1235-1242
Publication Date(Web):
DOI:10.1002/cbic.200700578
Abstract
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.
Co-reporter:Norihito Muranaka Dr.;Masanori Miura;Hikaru Taira Dr.
ChemBioChem 2007 Volume 8(Issue 14) pp:
Publication Date(Web):6 AUG 2007
DOI:10.1002/cbic.200700249
Expanding translation initiation. Incorporation of unnatural carboxylic acids without α-amino groups was achieved by using chemically acylated initiator tRNA (see figure). The results suggest that various unnatural compounds with a carboxyl group can be incorporated into the N terminus of proteins.
Co-reporter:Naoki Shozen, Takayoshi Watanabe, Takahiro Hohsaka
Journal of Bioscience and Bioengineering (June 2012) Volume 113(Issue 6) pp:704-709
Publication Date(Web):1 June 2012
DOI:10.1016/j.jbiosc.2012.01.018
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.
Co-reporter:Kim Phuong Huynh Nhat, Takayoshi Watanabe, Kensuke Yoshikoshi, Takahiro Hohsaka
Journal of Bioscience and Bioengineering (August 2016) Volume 122(Issue 2) pp:146-154
Publication Date(Web):1 August 2016
DOI:10.1016/j.jbiosc.2016.01.010
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.
Co-reporter:Yasunori Tokuda, Takayoshi Watanabe, Kazushi Horiike, Kaori Shiraga, ... Takahiro Hohsaka
Journal of Bioscience and Bioengineering (April 2011) Volume 111(Issue 4) pp:402-407
Publication Date(Web):1 April 2011
DOI:10.1016/j.jbiosc.2010.12.012
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.
