Article
Chemistry, Multidisciplinary
Xue-Yuan Wu, Meng-Yin Li, Shao-Jun Yang, Jie Jiang, Yi-Lun Ying, Peng R. Chen, Yi-Tao Long
Summary: Conventional methods for protein nanopore engineering are limited by the 20 natural amino acids, which restricts the diversity of nanopores. To overcome this, we used the genetic code expansion technique to incorporate unnatural amino acids into aerolysin nanopores. Molecular dynamics simulations and single-molecule sensing experiments showed that the newly incorporated amino acids provided a favorable environment for target molecule interactions. This work provides a new framework for creating nanopores with unique sensing properties.
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
(2023)
Article
Biochemistry & Molecular Biology
Hannae Lee, Dongchan Kim, Sooin Kim, Hyun Soo Lee
Summary: The study showed that converting UAAs into optically pure forms using two enzymes can increase protein yield in genetic code expansion. This method can be utilized for preparing optically pure UAAs and has broad substrate specificity for structurally diverse UAAs.
Article
Multidisciplinary Sciences
Hongxia Zhao, Wenlong Ding, Jia Zang, Yang Yang, Chao Liu, Linzhen Hu, Yulin Chen, Guanglong Liu, Yu Fang, Ying Yuan, Shixian Lin
Summary: This study describes the engineered chimeric phenylalanine systems that allow efficient incorporation of UAAs and the construction of E. coli strains dependent on UAAs for growth. The research findings suggest that synthetic auxotrophic cells can grow robustly in living mice.
NATURE COMMUNICATIONS
(2021)
Review
Biochemistry & Molecular Biology
Michiko Kimoto, Ichiro Hirao
Summary: This review discusses the current methods for incorporating new amino acids into proteins by designing new codon-anticodon interactions, including unnatural base pairs systems for expanding the genetic alphabet.
FRONTIERS IN MOLECULAR BIOSCIENCES
(2022)
Article
Biotechnology & Applied Microbiology
Eden Ozer, Lital Alfonta
Summary: Escherichia coli has been the most commonly used model bacterium in research for decades, but a faster synthetic biology chassis, Vibrio natriegens, is now emerging. Established methodologies in E. coli are being adapted for V. natriegens to create a faster platform for molecular biology studies. Genetic code expansion, such as incorporating unnatural amino acids into proteins, is a powerful tool for protein engineering and biorthogonal modifications, and has been successfully adapted for V. natriegens.
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
(2021)
Article
Biochemistry & Molecular Biology
Marcel Streit, Mareike Hemberger, Stephanie Haefner, Felix Knote, Tobias Langenhan, Gerti Beliu
Summary: The introduction of an engineered aminoacyl-tRNA synthetase/tRNA pair allows for the site-specific incorporation of unnatural amino acids into proteins. This method, called Genetic Code Expansion (GCE), can also be used to control the temporal incorporation of genetically encoded elements into proteins. In this study, an optimized GCE system (GCEXpress) was developed for efficient and fast incorporation of unnatural amino acids. The GCEXpress system was used to alter the subcellular localization of proteins and to study protein-protein interactions involved in immune functions and oncological processes. The results suggest that GCE, combined with biophysical measurements, is a valuable approach for analyzing the properties of proteins and their interactions with ligands.
Article
Biochemistry & Molecular Biology
Yusuke Kato
Summary: A protocol was developed for plasmid curing and exchange using a novel counter-selectable marker pylS(ZK)-pylT in Escherichia coli, which proved to be effective in both processes. The study demonstrated successful plasmid curing and exchange, highlighting the potential scalability and versatility of counter-selectable markers based on PylRS-tRNA(pyl).
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2021)
Article
Biochemistry & Molecular Biology
Li -Tao Guo, Kazuaki Amikura, Han -Kai Jiang, Takahito Mukai, Xian Fu, Yane-Shih Wang, Patrick O'Donoghue, Dieter Soell, Jeffery M. Tharp
Summary: The pyrrolysyl-tRNA synthetase (PylRS) is a major route to install noncanonical amino acids into proteins in living cells due to its tolerance for diverse amino acid substrates and orthogonality in multiple organisms. A novel class of PylRS enzymes was identified in a subset of methanogenic archaea, lacking the N-terminal tRNA-binding domain yet remaining active and orthogonal in bacteria and eukaryotes. Molecular phylogeny analysis revealed the coevolutionary history of PylRS and tRNAPyl, with the emergence of tRNAPyl sequences containing unique discriminator bases that may enable further genetic code expansion efforts.
JOURNAL OF BIOLOGICAL CHEMISTRY
(2022)
Article
Biochemistry & Molecular Biology
Daisuke Fuji, Takehiro Ando, Masashi Sato, Santhana Vedi, Yukio Takamori, Takumi Yokoyama, Mizuki Yamamoto, Takashi Kawakami
Summary: In this study, directed evolution was performed on human IL-5 using SELEX to discover novel IL-5-binding unnatural cyclic peptides. These peptides can be utilized in various research, therapeutic, and diagnostic applications involving IL-5 signaling.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(2022)
Article
Biochemistry & Molecular Biology
Natalie Elia
Summary: GFP revolutionized cell biology by enabling protein visualization in living cells, but newer imaging technologies face limitations due to size and photophysics of fluorescent proteins. An alternative approach of direct protein labeling with fluorescent dyes has been introduced, allowing for live-cell and super-resolution imaging without the need for protein-labeling tags.
Article
Biology
Lloyd Davis, Inja Radman, Angeliki Goutou, Ailish Tynan, Kieran Baxter, Zhiyan Xi, Jack M. O'Shea, Jason W. Chin, Sebastian Greiss
Summary: Researchers have developed a new genetic code expansion system that allows for selective switching on of genes in targeted neurons using light activation, revealing the distinct contributions and synergistic roles of individual neurons in animal behavior.
Article
Biochemical Research Methods
Arnaz Ranji Charna, Benjamin J. Des Soye, Ioanni Ntai, Neil L. Kelleher, Michael C. Jewett
Summary: Incorporation of noncanonical amino acids into proteins using a highly efficient cell-free protein synthesis platform has been reported in this study. This platform enables the site-specific incorporation of Pyrrolysine-based noncanonical amino acids into proteins at high suppression efficiency.
BIOTECHNOLOGY JOURNAL
(2022)
Article
Biochemical Research Methods
Xinyuan He, Tianyu Gao, Yan Chen, Kun Liu, Jiantao Guo, Wei Niu
Summary: In this study, we successfully achieved genetic code expansion in Pseudomonas putida KT2440, introducing non-natural amino acids into proteins. By optimizing the decoding system, we successfully incorporated four non-natural amino acids into proteins and demonstrated the utility of genetic code expansion in protein-protein interaction studies.
ACS SYNTHETIC BIOLOGY
(2022)
Article
Chemistry, Physical
Alejandro Gran-Scheuch, Elisa Bonandi, Ivana Drienovska
Summary: This study reports the design, synthesis, and characterization of non-canonical amino acids (ncAAs) inspired by organocatalysts, which were successfully incorporated into proteins. The incorporation of these ncAAs expands the toolbox for protein engineering and chemical biology applications.
Review
Biochemistry & Molecular Biology
Mayilvahanan Aarthy, Augustine George, Niraikulam Ayyadurai
Summary: Fluorescent proteins have been engineered to incorporate unnatural amino acids, expanding their applications in various biological fields by altering their properties.
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
(2021)
Article
Biochemistry & Molecular Biology
Qingke Li, Qu Chen, Paul C. Klauser, Mengyuan Li, Feng Zheng, Nanxi Wang, Xiaoying Li, Qianbing Zhang, Xuemei Fu, Qian Wang, Yang Xu, Lei Wang
Article
Chemistry, Multidisciplinary
Xin X. Zhou, Colton J. Bracken, Kaihua Zhang, Jie Zhou, Yun Mou, Lei Wang, Yifan Cheng, Kevin K. Leung, James A. Wells
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
(2020)
Article
Biochemistry & Molecular Biology
Viktoriya Y. Berdan, Paul C. Klauser, Lei Wang
Summary: Drugs with a covalent mechanism of action have advantages in potency, selectivity, and in vivo efficacy. While traditionally limited to small molecules, recent advancements in peptide and protein therapeutics have allowed for the targeting of previously undruggable proteins and protein-protein interactions using covalent mechanisms.
BIOORGANIC & MEDICINAL CHEMISTRY
(2021)
Article
Biochemistry & Molecular Biology
Li Cao, Jun Liu, Farid Ghelichkhani, Sharon Rozovsky, Lei Wang
Summary: Researchers have developed orthogonal tRNA(Pyl)-MaBzKRS pairs for site-specific modification of proteins, allowing for efficient incorporation of epsilon-N-benzoyllysine (BzK) into proteins in E. coli and mammalian cells. MaBzKRS variants with mutations at different positions in the amino acid binding pocket have been identified for incorporating BzK, which could have broad utility in studying the biological effects of lysine benzoylation.
Article
Chemistry, Multidisciplinary
Jun Liu, Li Cao, Paul C. Klauser, Rujin Cheng, Viktoriya Y. Berdan, Wei Sun, Nanxi Wang, Farid Ghelichkhani, Bingchen Yu, Sharon Rozovsky, Lei Wang
Summary: Introducing novel chemical bonds into proteins offers innovative possibilities, with the incorporation of latent bioreactive unnatural amino acids providing a powerful system for covalent targeting of natural residues. This approach has potential applications in biochemical research and protein engineering.
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
(2021)
Review
Biochemistry & Molecular Biology
Nanxi Wang, Lei Wang
Summary: By incorporating latent bioreactive amino acids into proteins through genetic code expansion, selective covalent linkages can be generated, enabling the development of covalent protein drugs with improved therapeutic potential.
CURRENT OPINION IN CHEMICAL BIOLOGY
(2022)
Review
Biochemistry & Molecular Biology
Li Cao, Lei Wang
Summary: By designing latent bioreactive unnatural amino acids and genetically encoding them into proteins, the covalent bonding ability of proteins can be expanded, creating new covalent bonds within and between proteins. These bonds enhance protein properties, modulate protein function, probe protein interactions, and develop covalent protein drugs.
Article
Chemistry, Multidisciplinary
Bingchen Yu, Shanshan Li, Takako Tabata, Nanxi Wang, Li Cao, G. Renuka Kumar, Wei Sun, Jun Liu, Melanie Ott, Lei Wang
Summary: This study developed covalent nanobodies that bind irreversibly with SARS-CoV-2, increasing neutralization potency against wild-type virus and its variants. These insights into increased potency can be valuable for developing effective therapeutics against viral infections.
Article
Chemistry, Multidisciplinary
Shanshan Li, Nanxi Wang, Bingchen Yu, Wei Sun, Lei Wang
Summary: Protein-carbohydrate interactions are important in biological processes but challenging to study. This study engineered covalent linkages between proteins and carbohydrates, offering a solution to overcome the low affinity and weak strength of these interactions.
Article
Chemistry, Multidisciplinary
Wei Sun, Nanxi Wang, Hongjiang Liu, Bingchen Yu, Ling Jin, Xingjie Ren, Yin Shen, Lei Wang
Summary: Protein-RNA interactions play a crucial role in regulating the fate and function of RNA. This study demonstrates a novel approach, using genetically encoded unnatural amino acids, to crosslink proteins with bound RNA and analyze their interactions. The technique allows for the identification of specific RNA molecules and the discovery of unknown chemical modifications in cells. This research is highly significant in advancing our understanding of protein-RNA interactions in biological processes.
Review
Biochemistry & Molecular Biology
Jun Liu, Bing Yang, Lei Wang
Summary: Photo- and chemical crosslinking of proteins have provided diverse approaches for studying protein structure and interactions with biomolecules. Recent advancements in the development of photoactivatable groups and selective reactivity have improved crosslinking efficiency and identification. The use of residue selective chemical functional groups, activated by light or proximity, in small molecule crosslinkers and genetically encoded unnatural amino acids is summarized. Together with new software development for crosslink identification, residue selective crosslinking has enhanced the research of elusive protein-protein interactions in vitro, cell lysate, and live cells. Residue selective crosslinking is expected to expand to other methods for investigating various protein-biomolecule interactions.
CURRENT OPINION IN CHEMICAL BIOLOGY
(2023)
Article
Chemistry, Multidisciplinary
Paul C. Klauser, Shalini Chopra, Li Cao, Kondapa Naidu Bobba, Bingchen Yu, Youngho Seo, Emily Chan, Robert R. Flavell, Michael J. Evans, Lei Wang
Summary: By binding irreversibly to the target, covalent nanobodies increase the radioactive dose to the tumor without altering the pharmacokinetics of the drug or affecting normal tissue distribution. This covalent approach improves tumor responses to targeted radionuclide therapies and can be applied to various protein radiopharmaceuticals for treating a wide range of tumors.
ACS CENTRAL SCIENCE
(2023)
Article
Chemistry, Multidisciplinary
Bingchen Yu, Li Cao, Shanshan Li, Paul C. Klauser, Lei Wang
Summary: The proximity-enabled sulfur(vi) fluoride exchange (SuFEx) reaction is capable of generating specific covalent linkages between proteins in cells and in vivo, which provides new avenues for studying elusive protein-protein interactions and developing potent covalent protein drugs. This study systematically investigated the kinetics of SuFEx reaction in different proteins and conditions, revealing a two-step mechanism involving noncovalent binding followed by covalent bond formation. The results have important implications for the exploration of SuFEx-enabled covalent proteins in basic biological research and innovative biotherapeutics.
Article
Chemistry, Multidisciplinary
Paul C. Klauser, Viktoriya Y. Berdan, Li Cao, Lei Wang
Summary: The introduction of new covalent bonds into proteins has provided novel avenues for protein research and applications, but generating covalent linkages remains challenging. In this study, genetically encoded mFSY selectively reacted with specific amino acids, allowing successful engineering of various proteins into covalent binders.
CHEMICAL COMMUNICATIONS
(2022)
Article
Materials Science, Biomaterials
Chao Liu, Ting Wu, Xin Shu, Shang-Tong Li, Daniel R. Wang, Nanxi Wang, Rong Zhou, Hao Yang, Hong Jiang, Ivo A. Hendriks, Pengyun Gong, Long Zhang, Michael L. Nielsen, Kui Li, Lei Wang, Bing Yang
Summary: The software OpenUaa is developed for identifying protein crosslinks generated by genetically encoded unnatural amino acids and endogenous protein conjugation, dramatically improving identification sensitivity and coverage. Integrating GECX with OpenUaa enabled the successful identification of the direct interactome of thioredoxin in Escherichia coli cells and significantly improved the coverage of SUMOylated peptides.
