Article
Multidisciplinary Sciences
Laura Grasemann, Barbora Lavickova, M. Carolina Elizondo-Cantu, Sebastian J. Maerkl
Summary: The PURE system is a promising chassis for cell-free synthetic biology, but commercially available systems are expensive while homemade methods are time-consuming and laborious. Streamlining protein purification through coculturing and co-purification can minimize time and labor requirements.
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
(2021)
Article
Biochemistry & Molecular Biology
Nikolay A. Aleksashin, Stacey Tsai-Lan Chang, Jamie H. D. Cate
Summary: This article presents an optimized human cell-free translation system that simplifies lysate preparation and maintains low levels of eIF2 alpha phosphorylation. The system overexpresses GADD34 and K3L proteins, which suppress phosphorylation of eIF2 alpha, to bypass the limitations of currently used in vitro systems. This new CFPS system is valuable for studying human translation mechanisms in more physiological conditions.
Article
Biochemical Research Methods
Sean S. Chung, Erik J. Bidstrup, Jasmine M. Hershewe, Katherine F. Warfel, Michael C. Jewett, Matthew P. DeLisa
Summary: A method for displaying and evolving proteins with complex post-translational modifications has been developed, allowing for the efficient and site-specific modification of target proteins with different glycan structures.
ACS SYNTHETIC BIOLOGY
(2022)
Review
Cell Biology
Marianne Mercer, Seoyeon Jang, Chunyang Ni, Michael Buszczak
Summary: Regulation of mRNA translation is crucial for germ cell development and function across species, involving specific RNA binding proteins. These proteins are conserved and play similar roles in germ cells of various species, highlighting the importance of understanding the mechanisms controlling mRNA translation in germ cells.
FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY
(2021)
Article
Biochemistry & Molecular Biology
Daniel H. Goldman, Nathan M. Livingston, Jonathan Movsik, Bin Wu, Rachel Green
Summary: Translation of problematic mRNA sequences induces ribosome stalling, with slow ribosome rescue allowing cells to distinguish between transient and deleterious stalls. The dynamics of ribosome collisions and their recognition facilitate selective targeting for quality control.
Article
Biochemistry & Molecular Biology
Ayako Yamakawa, Tatsuya Niwa, Yuhei Chadani, Akinao Kobo, Hideki Taguchi
Summary: Life depends on proteins, and recent advances have shown that nascent chains (pep-tRNAs) directly participate in various cell functions. This study presents a new method called PETEOS to enrich pep-tRNAs and identify their polypeptide moieties. Using Escherichia coli as a model, around 800 proteins were identified, with a bias towards the N-termini, reflecting the population of nascent chains captured by PETEOS. This method can complement conventional approaches in investigating nascent chains in cells.
NUCLEIC ACIDS RESEARCH
(2023)
Article
Plant Sciences
Kasia Dinkeloo, Zoe Pelly, John M. McDowell, Guillaume Pilot
Summary: Translating ribosome affinity purification (TRAP) is a method that uses transgenic plants to purify ribosomes and translating mRNAs. It can be used for studying actively translated mRNAs by quantitative PCR or RNA sequencing. This approach allows researchers to isolate the translatome of specific cell types using condition- or cell-specific promoters. However, it may lack sensitivity in cases where the condition/cell-specific promoter is weak, ribosome turnover is low, or the targeted cells are short-lived. To overcome these limitations, a new TRAP system was developed that utilizes two transgenes and provides increased sensitivity and better temporal resolution.
Article
Hematology
Jan-Erik Messling, Karl Agger, Kasper L. Andersen, Kristina Kromer, Hanna M. Kuepper, Anders H. Lund, Kristian Helin
Summary: Novel therapies are urgently needed for the treatment of acute myeloid leukemia (AML). A study identified RIOK2 as a potential target for AML treatment, as its loss leads to decreased protein synthesis and apoptosis in leukemic cells.
Article
Biochemistry & Molecular Biology
Sonja Zayni, Samar Damiati, Susana Moreno-Flores, Fabian Amman, Ivo Hofacker, David Jin, Eva-Kathrin Ehmoser
Summary: Membrane proteins play important roles in cellular biology and are key drug targets. Progress in immune effector cell therapy has expanded the use of synthetic membrane receptors, but studying membrane proteins is challenging due to difficulties in vitro synthesis. By utilizing cell-free synthesis and optimizing plasmid sequences, researchers are able to better understand and improve the expression efficiency of membrane proteins like VDAC for drug-based therapy.
Article
Biochemistry & Molecular Biology
Gaetano D'Urso, Charlotte Guyomar, Sophie Chat, Emmanuel Giudice, Reynald Gillet
Summary: The bacterial trans-translation quality control system is responsible for rescuing stalled ribosomes on mRNA lacking a stop codon. This system involves the synergistic action of two partners, tmRNA and SmpB, to monitor and intervene in protein synthesis.
Article
Medicine, Research & Experimental
Florian Heinkel, Meghan M. Verstraete, Siran Cao, Janessa Li, Patrick Farber, Elizabeth Stangle, Begonia Silva-Moreno, Fangni Peng, Surjit Dixit, Martin J. Boulanger, Thomas Spreter Von Kreudenstein, Eric Escobar-Cabrera
Summary: The newly developed heterodimeric IgA Fc platform allows for the development of novel, multifunctional, and multimeric molecules that have the potential to transform the next generation of antibody therapeutics.
Article
Multidisciplinary Sciences
Wakana Sato, Judee Sharon, Christopher Deich, Nathaniel Gaut, Brock Cash, Aaron E. Engelhart, Katarzyna P. Adamala
Summary: Cell-free protein expression is widely used in various fields. Escherichia coli (E. coli) is the most popular cell-free system. In this study, we created a RecB knockout strain of E. coli, named Akaby, which is optimized for cell-free expression. Our results showed that Akaby successfully reduced linear DNA degradations and improved protein expression efficiency. Akaby is an efficient and simple alternative for linear template expression in cell-free reactions.
Review
Multidisciplinary Sciences
Yaeta Endo
Summary: The review discusses the limitations of conventional cell-free protein synthesis systems and how these have been addressed through the development of a new system based on wheat embryos, offering high translation activity and robustness for research and applied sciences.
PROCEEDINGS OF THE JAPAN ACADEMY SERIES B-PHYSICAL AND BIOLOGICAL SCIENCES
(2021)
Review
Biochemical Research Methods
Harini Sridharan, Fernanda Piorino, Mark P. Styczynski
Summary: Cell-free expression systems are widely used in biotechnology, but many aspects of these systems are still poorly understood. Systems biology studies have provided insights into the characterization of cell-free proteome, metabolome, and their relationship to system performance. Promising future research directions are highlighted.
CURRENT OPINION IN BIOTECHNOLOGY
(2022)
Article
Biochemical Research Methods
Taylor Sheahan, Hans-Joachim Wieden
Summary: Cell-free expression systems hold great promise for engineering biological and life-similar systems. However, the protein yields are limited by the constraints of ribosome and ribosome-associated processes. This study shows that adding purified recombinant S1 can double the protein yields in a commercial in vitro reconstituted TX-TL system.
ACS SYNTHETIC BIOLOGY
(2022)
Article
Biochemistry & Molecular Biology
Yusuke Uwaguchi, Kei Fujiwara, Nobuhide Doi
Summary: A new method of GUV fusion using a divalent cation and long chain polyethylene glycol was reported, showing significant fusion without leakage and maintaining cell-free transcription-translation ability inside GUVs without external supplementation of macromolecules. This method can be applied for switching ON transcription-translation in GUVs in a fusion-dependent manner, extending bottom-up synthetic biology and molecular robotics that use GUVs as a chassis.
Article
Biology
Katsuki Takahashi, Gaku Sato, Nobuhide Doi, Kei Fujiwara
Summary: The study found that high concentration NTP can restore the activity of CFPS using high concentration cell extracts; The NTP restoration is independent and NTP derivatives can also improve CFPS levels; Dialysis mode can reduce the NTP requirement and increase the reaction speed of CFPS using high concentration cell extracts.
Article
Multidisciplinary Sciences
Toshiki Akui, Kei Fujiwara, Gaku Sato, Masahiro Takinoue, Shin-ichiro M. Nomura, Nobuhide Doi
Summary: Biochemical systems in living cells have an optimal concentration ratio among each constituent element to maintain functionality. However, in systems with complex interactions and feedbacks, the concentration shift of the entire system can greatly change its activity. This study uses a TX-TL system to illustrate the nonlinear relationship between system concentration and activity, showing how concentration shifts can act as a switch for system activity and influence its function.
Article
Biochemistry & Molecular Biology
Mayuko Suzuki, Kouta Iwaki, Moeki Kikuchi, Kei Fujiwara, Nobuhide Doi
Summary: The study found that the fusogenic peptide S19 enhances the endosomal escape efficiency of TAT-fused proteins. Mutagenesis analysis showed that the amino acids with high beta-sheet forming propensities in S19 are important for the intracellular uptake of S19-TAT fusion proteins. The appropriate linking position and order of S19 and TAT peptides were also investigated, and both the previous C-terminal S19-TAT tag and the N-terminal TAT-S19 tag promote the cytoplasmic delivery of the fusion protein.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(2022)
Article
Multidisciplinary Sciences
Sakura Takada, Natsuhiko Yoshinaga, Nobuhide Doi, Kei Fujiwara
Summary: Through studying the mode selection mechanism of Min waves, it was found that the balance of MinD's binding and dissociation from the membrane plays a crucial role in mode selection, and hysteresis characteristics were observed in the transition of wave modes.
Article
Chemistry, Multidisciplinary
Sakura Takada, Natsuhiko Yoshinaga, Nobuhide Doi, Kei Fujiwara
Summary: Reaction-diffusion waves are self-organization structures generated by nanosize molecules and play a fundamental role in patterning in materials and spatiotemporal regulations in living cells. We developed an artificial cell system with tunable period of reaction-diffusion waves and established an important framework for constructing molecular robots with spatiotemporal units.
Article
Chemistry, Multidisciplinary
Miho Yanagisawa, Chiho Watanabe, Natsuhiko Yoshinaga, Kei Fujiwara
Summary: Polymer micromaterials covered with a surfactant membrane are widely used in pharmaceuticals, cosmetics, and foods. This study analyzes the cell-size space effect (CSE) on molecular diffusion, nanostructure transition, and phase separation, and identifies factors such as short-and long-range interactions with the membrane surface and small volume that contribute to CSE. The study also explores other factors of CSE through the analysis of protein reaction-diffusion systems and biochemical reactions.
Article
Chemistry, Multidisciplinary
Saki Nishikawa, Gaku Sato, Sakura Takada, Shunshi Kohyama, Gen Honda, Miho Yanagisawa, Yutaka Hori, Nobuhide Doi, Natsuhiko Yoshinaga, Kei Fujiwara
Summary: This study demonstrates that the co-existence of various macromolecules in cell-size space can regulate protein localization and biochemical reaction systems. Experimental and theoretical analyses show that a large surface-area-to-volume ratio enhances membrane localization of molecules, while competitive binding to lipid membranes among multiple proteins alleviates interfacial effects.
Article
Biochemical Research Methods
Yukino Matsui, Toshiki Akui, Nobuhide Doi, Kei Fujiwara
Summary: A method for activating protein synthesis in liposomes encapsulating cell extract-based systems by hypertonic concentration is presented. This protocol can be used for developing artificial cells that activate protein expression in response to external stimuli.
Article
Chemistry, Multidisciplinary
Tatsuki Deyama, Yukino Matsui, Yuhei Chadani, Yasuhiko Sekine, Nobuhide Doi, Kei Fujiwara
Summary: Researchers have successfully created artificial cells where the genome information of living cells is expressed by encoded elements. They have confirmed that the system functions normally within the artificial cells, which opens up possibilities for reconstructing living cells from biomolecules.
CHEMICAL COMMUNICATIONS
(2021)
