Article
Chemistry, Multidisciplinary
Liya Ye, Xianlu Lei, Liguang Xu, Hua Kuang, Chuanlai Xu, Xinxin Xu
Summary: The prevalent Omicron variant of SARS-CoV-2 has high infectivity and strong concealment, resulting in more asymptomatic infections. Antigen detection is a simple, fast, and convenient method that can supplement nucleic acid detection for early detection and real-time control of infection sources. This study developed a gold nanoparticle-based immunochromatographic assay using a recombinant nucleocapsid protein derived from Omicron, which showed high efficiency in detecting Omicron sub-lineages and had no cross-reactivity with human coronaviruses.
MATERIALS CHEMISTRY FRONTIERS
(2023)
Article
Entomology
Takeru Ebihara, Jian Xu, Yoshino Tonooka, Takumi Nagasato, Kohei Kakino, Akitsu Masuda, Kosuke Minamihata, Noriho Kamiya, Hirokazu Nakatake, Yuuka Chieda, Hiroaki Mon, Tsuguru Fujii, Takahiro Kusakabe, Jae Man Lee
Summary: The study successfully expressed and purified human and murine TNF alpha proteins using silkworm-BEVS, demonstrating ideal productivity with similar biological activity. It was found that C-terminal fusion tags negatively impacted the biological activity of TNF alpha, highlighting the importance of tag removal for improved cytokine activity.
Article
Biochemistry & Molecular Biology
Minttu S. Virolainen, Cecilie L. Soltoft, Per A. Pedersen, Lars Ellgaard
Summary: The research team successfully produced full-length FICD by combining a yeast platform and GFP tagging, providing a new approach for optimizing conditions for expression, solubilization, and purification. They also highlighted that this method is not only applicable to FICD, but also to other membrane proteins.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2022)
Article
Biochemistry & Molecular Biology
Michele Galluccio, Tiziano Mazza, Mariafrancesca Scalise, Maria Chiara Sarubbi, Cesare Indiveri
Summary: This study successfully expressed the carnitine transporter CT2 in Escherichia coli and found that the recombinant protein is capable of transporting carnitine. These findings have significant implications for the basic research on membrane transporters and their applications in human health.
MOLECULAR BIOLOGY REPORTS
(2022)
Article
Multidisciplinary Sciences
Robert M. Cooper, Taishi Tonooka, Andriy Didovyk, Jeff Hasty
Summary: This method describes a cost-effective and time-efficient way to produce bacterial cell-free lysate supporting high levels of gene expression, with the potential for dynamic gene circuits through heterologous expression of the ClpX protease. It is suitable for high-sensitivity biosensing applications.
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
(2021)
Article
Biochemical Research Methods
Yajie Tang, Shengming Ma, Sen Lin, Yinrong Wu, Siyang Chen, Gang Liu, Lisong Ma, Zaihua Wang, Lele Jiang, Yao Wang
Summary: In this study, high-quality soluble CD1E was successfully synthesized using an E.coli cell-free protein synthesis system, and it was purified with >= 90% purity by Ni2+ affinity chromatography. The protein interaction network of CD1E was predicted using the string website, and a potential binding partner B2M was identified. The interaction between CD1E and B2M was verified using Surface Plasmon Resonance (SPR).
PROTEIN EXPRESSION AND PURIFICATION
(2023)
Article
Cell Biology
Nathan M. Fastman, Yuxi Liu, Vyas Ramanan, Hanne Merritt, Eileen Ambing, Anna A. DePaoli-Roach, Peter J. Roach, Thomas D. Hurley, Kevin T. Mellem, Julie C. Ullman, Eric Green, Jr. David Morgans, Christos Tzitzilonis
Summary: This study reveals the structure of the human GYS1:GYG1 complex in different functional states, providing insights into the mechanism of glycogen synthesis. The asymmetric conformation of GYS1 exposes an interface for close association with GYG1, facilitating the handoff and elongation of the glycogen chain. Full activation of GYS1 widens the GYG1-binding groove, allowing for GYG1 release and concomitant glycogen chain growth. This structural mechanism explains the stepwise nature of glycogen synthesis and suggests potential targets for therapeutic interventions in glycogen storage diseases.
Article
Multidisciplinary Sciences
Krishna Kumar Haridhasapavalan, Pradeep Kumar Sundaravadivelu, Neha Joshi, Nayan Jyoti Das, Anshuman Mohapatra, Udayashree Voorkara, Vishwas Kaveeshwar, Rajkumar P. Thummer
Summary: This study reports the generation of recombinant human HAND2 fusion protein using a heterologous system. The purified protein retained its secondary structure and could transduce cells and translocate to the nucleus. Additionally, the recombinant protein showed angiogenic potential and synergistically activated the α-MHC promoter.
SCIENTIFIC REPORTS
(2022)
Article
Biochemistry & Molecular Biology
Jasmin Hertler, Kaouthar Slama, Benedikt Schober, Zeynep Ozrendeci, Virginie Marchand, Yuri Motorin, Mark Helm
Summary: Techniques have been developed to isolate long, translatable, and internally point-modified mRNA, enabling functional analysis of these modified mRNAs.
NUCLEIC ACIDS RESEARCH
(2022)
Article
Agriculture, Multidisciplinary
Junjian Ran, Lingxia Jiao, Ruixiang Zhao, Mingming Zhu, Jianrong Shi, Baocheng Xu, Li Pan
Summary: This study successfully isolated an antagonistic bacteria strain P. polymyxa 7F1 with antifungal activity and purified a novel antifungal protein from it, showing broad-spectrum antifungal activity. The recombinant protein demonstrated similar antifungal activity to the purified protein, providing an effective way to control Fusarium head blight.
JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE
(2021)
Article
Biochemical Research Methods
Madison A. DeWinter, Ariel Helms Thames, Laura Guerrero, Weston Kightlinger, Ashty S. Karim, Michael C. Jewett
Summary: In resource-limited settings, point-of-care drug manufacturing using cell-free protein synthesis (CFPS) and affinity purification can overcome cold chain and infrastructure constraints. By synthesizing peptide hormones locally on-demand, this platform shows potential for decentralized production of valuable drugs.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Biochemical Research Methods
Madison A. DeWinter, Ariel Helms Thames, Laura Guerrero, Weston Kightlinger, Ashty S. Karim, Michael C. Jewett
Summary: In resource-limited settings, point-of-care drug manufacturing using cell-free protein synthesis (CFPS) and affinity purification and enzymatic cleavage scheme could overcome the challenges of delivering sensitive biologic medicines. This platform was successfully used to synthesize peptide hormones which are used to treat various diseases. With further development, it has the potential to revolutionize the decentralized manufacturing of peptide hormone drugs.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Biotechnology & Applied Microbiology
Chandrima Dey, Madhuri Thool, Srirupa Bhattacharyya, S. Sudhagar, Rajkumar P. Thummer
Summary: The study described successful heterologous expression and purification of human OCT4 protein in E. coli, with confirmation of its function in cells. The recombinant protein can be used for derivation of integration-free pluripotent cells and other biological research purposes.
Article
Biochemistry & Molecular Biology
Marina Schramm, Stephanie Friedrich, Kai-Uwe Schmidtke, Jan Kiebist, Paul Panzer, Harald Kellner, Rene Ullrich, Martin Hofrichter, Katrin Scheibner
Summary: In this study, cell-free protein synthesis using lysates from filamentous fungi was used as a third approach for the expression of unspecific peroxygenases (UPOs). The expressed enzyme showed comparable activities and catalytic performance to the wild-type enzyme, suggesting that cell-free expression could be a valuable tool for accessing and expanding the spectrum of UPO biocatalysts.
Article
Multidisciplinary Sciences
Ariel J. Ben-Sasson, Joseph L. Watson, William Sheffler, Matthew Camp Johnson, Alice Bittleston, Logeshwaran Somasundaram, Justin Decarreau, Fang Jiao, Jiajun Chen, Ioanna Mela, Andrew A. Drabek, Sanchez M. Jarrett, Stephen C. Blacklow, Clemens F. Kaminski, Greg L. Hura, James J. De Yoreo, Justin M. Kollman, Hannele Ruohola-Baker, Emmanuel Derivery, David Baker
Summary: By designing rigid interfaces, we have successfully generated binary layers co-assembled by two protein components, and experiments have shown that this material can rapidly form ordered structures in vitro and in cells. This design approach allows for customization and symmetry reconfiguration, enabling the formation of ligand arrays that can drive receptor clustering, downstream protein recruitment, and signaling.
Article
Multidisciplinary Sciences
Mathias Nielsen, Noortje de Haan, Weston Kightlinger, Zilu Ye, Sally Dabelsteen, Minyan Li, Michael C. Jewett, Ieva Bagdonaite, Sergey Y. Vakhrushev, Hans H. Wandall
Summary: This study systematically analyzed the isoform-specific targets of all GalNAc-Ts expressed in skin cells, revealing the biological effects of O-glycan initiation on epithelial formation. Over 300 unique glycosylation sites were identified on a diverse set of proteins, regulated by specific GalNAc-T isoforms, highlighting their impact on tissue phenotypes. The variability in O-glycosylation site-occupancy of secreted proteins suggests the importance of individual O-glycosylation sites in driving biological functions.
NATURE COMMUNICATIONS
(2022)
News Item
Biochemistry & Molecular Biology
Grant M. Landwehr, Michael C. Jewett
Summary: Inspired by nature, a synthetic carbon fixation cycle is able to build complex molecules directly from CO2. Several innovative advancements are required to build metabolism from the ground up, and now a strategy to balance carbon demands in a complex metabolic network is being explored.
NATURE CHEMICAL BIOLOGY
(2023)
Article
Biochemical Research Methods
Katherine F. Warfel, Asher Williams, Derek A. Wong, Sarah E. Sobol, Primit Desai, Jie Li, Yung-Fu Chang, Matthew P. DeLisa, Ashty S. Karim, Michael C. Jewett
Summary: In this study, maltodextrin is identified as an additive to cell-free reactions that can increase thermostability and serve as a low-cost energy substrate. The optimized formulations of conjugate vaccines demonstrate storage stability at different temperatures and generate bactericidal antibodies against enterotoxigenic Escherichia coli (ETEC) O78 O-polysaccharide. This low-cost, thermostable cell-free glycoprotein synthesis system has the potential to revolutionize medicine biosynthesis and distribution by bypassing cold-chain requirements.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Biochemical Research Methods
Blake J. Rasor, Payal Chirania, Grant A. Rybnicky, Richard J. Giannone, Nancy L. Engle, Timothy J. Tschaplinski, Ashty S. Karim, Robert L. Hettich, Michael C. Jewett
Summary: Researchers investigated the complexity of proteins and metabolites in Escherichia coli cell-free reactions, revealing the impact of cold shock-like proteins and buffer composition on gene expression.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Biochemistry & Molecular Biology
Kosuke Seki, Joey L. Galindo, Ashty S. Karim, Michael C. Jewett
Summary: Non-canonical amino acids (ncAAs) can be incorporated into peptides and proteins to create new properties and functions. In this study, a cell-free gene expression system based on Escherichia coli crude extract was developed to rapidly express and characterize functional suppressor tRNAs. The system co-expresses orthogonal tRNAs using endogenous machinery alongside a reporter protein, allowing for easy evaluation of suppression activity. This cell-free workflow can also be used to discover new suppressor tRNAs in metagenomic data. Overall, this system has the potential to accelerate the development of orthogonal translation systems for synthetic biology.
ACS CHEMICAL BIOLOGY
(2023)
Article
Biochemical Research Methods
Jessica C. Stark, Thapakorn Jaroentomeechai, Katherine F. Warfel, Jasmine M. Hershewe, Matthew P. DeLisa, Michael C. Jewett
Summary: The authors present a protocol for generating bacterial cell lysates and freeze-dried, cell-free reactions for the synthesis of glycoprotein therapeutics and vaccines. This method addresses the limitation of distributed biomanufacturing platforms in producing robust amounts of glycoproteins, which are critical in biologic development. The protocol allows for rapid production of desired glycoproteins and can be completed within one week. The authors anticipate that these cell-free technologies, along with the comprehensive user manual, will accelerate the development and distribution of glycoprotein therapeutics and vaccines.
Article
Multidisciplinary Sciences
Antje M. Krueger, Andrew Watkins, Roger Wellington-Oguri, Jonathan Romano, Camila Kofman, Alysse DeFoe, Yejun Kim, Jeff Anderson-Lee, Eli Fisker, Jill E. Townley, Anne d'Aquino, Rhiju C. Das, Michael Jewett
Summary: By integrating community science and experimental screening, the authors have developed a method for rational design of ribosomes with beneficial properties. They use an online video game, Eterna, to crowdsource RNA sequence design and apply their framework to discover mutant rRNA sequences that improve protein synthesis and cell growth. This work provides insights into rRNA sequence-function relationships and has implications for synthetic biology.
NATURE COMMUNICATIONS
(2023)
Article
Biochemical Research Methods
Ariel Helms Thames, Sam J. Moons, Derek A. Wong, Thomas J. Boltje, Bruce S. Bochner, Michael C. Jewett
Summary: The study developed a cell-free glycoprotein synthesis platform called GlycoCAP for building noncanonical glycans. Through optimization, the synthesis achieved over 60% sialylation efficiency with a noncanonical azido-sialic acid. The study also demonstrated the conjugation of a model fluorophore using click chemistry. GlycoCAP is expected to facilitate the development and discovery of glycan-based drugs.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Biochemical Research Methods
Madison A. DeWinter, Ariel Helms Thames, Laura Guerrero, Weston Kightlinger, Ashty S. Karim, Michael C. Jewett
Summary: In resource-limited settings, point-of-care drug manufacturing using cell-free protein synthesis (CFPS) and affinity purification can overcome cold chain and infrastructure constraints. By synthesizing peptide hormones locally on-demand, this platform shows potential for decentralized production of valuable drugs.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Biochemical Research Methods
Blake J. Rasor, Ashty S. Karim, Hal S. Alper, Michael C. Jewett
Summary: Cell-free synthetic biology allows for rapid prototyping and synthesis of biological parts and products without the constraints of cell growth. However, the variability in composition and activity of cell extracts can make them unpredictable for practical laboratory use. This study found that cell extracts from Escherichia coli and Saccharomyces cerevisiae retain consistent metabolic activity after long-term storage and multiple freeze-thaw cycles, providing valuable insight into the behavior of stored extracts in cell-free systems.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Biochemistry & Molecular Biology
Asher J. J. Williams, Katherine F. F. Warfel, Primit Desai, Jie Li, Jen-Jie Lee, Derek A. A. Wong, Phuong M. M. Nguyen, Yufan Qin, Sarah E. E. Sobol, Michael C. C. Jewett, Yung-Fu Chang, Matthew P. P. DeLisa
Summary: Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal disease and death worldwide, especially in infants and young children. However, the development of an affordable vaccine to reduce mortality and morbidity associated with ETEC infection in children under the age of 5 years remains an unmet goal. In this study, we describe a cost-effective method to produce conjugate vaccine candidates against prevalent O serogroups of ETEC using glycoengineered strains of non-pathogenic E. coli. The resulting conjugates stimulate strong immune responses and provide evidence of vaccine-induced mucosal protection in mice.
FRONTIERS IN MOLECULAR BIOSCIENCES
(2023)
Article
Multidisciplinary Sciences
Andrew C. Hunt, Bastian Voegeli, Ahmed O. Hassan, Laura Guerrero, Weston Kightlinger, Danielle J. Yoesep, Antje Krueger, Madison DeWinter, Michael S. Diamond, Ashty S. Karim, Michael C. Jewett
Summary: The authors developed a workflow using cell-free protein synthesis to express and evaluate hundreds of antibody fragments in less than 24 hours. They applied this workflow to evaluate antibodies targeting SARS-CoV-2 and identified potent neutralizing antibody candidates. This cell-free workflow is expected to accelerate antibody discovery for future pandemics and various applications.
NATURE COMMUNICATIONS
(2023)
Article
Biochemical Research Methods
Ariel Helms Thames, Clayton H. Rische, Yun Cao, Rebecca A. Krier-Burris, Fei Li Kuang, Robert G. Hamilton, Charles Bronzert, Bruce S. Bochner, Michael C. Jewett
Summary: Allergens are utilized in clinical settings for the diagnosis and treatment of allergic diseases. A cell-free protein synthesis approach has been demonstrated for the production of allergens, enabling the recognition of allergens by allergen-specific immunoglobulin E (IgE) and activation of human cells in an IgE-dependent manner. This platform has the potential to provide diagnostic and therapeutic technologies for allergists and allergic patients.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Biochemistry & Molecular Biology
Katherine F. Warfel, Eugenie Laigre, Sarah E. Sobol, Emilie Gillon, Annabelle Varrot, Olivier Renaudet, Jerome Dejeu, Michael C. Jewett, Anne Imberty
Summary: Researchers have developed a new method for efficient expression of multivalent, disulfide bond-rich, rhamnose-binding lectins. This method allows for rapid expression and characterization without purification. The workflow enables determination of lectin substrate specificity and binding affinity. This method facilitates high-throughput expression, screening, and characterization of new and engineered multivalent lectins for synthetic glycobiology applications.
Article
Biochemical Research Methods
Madison A. DeWinter, Ariel Helms Thames, Laura Guerrero, Weston Kightlinger, Ashty S. Karim, Michael C. Jewett
Summary: In resource-limited settings, point-of-care drug manufacturing using cell-free protein synthesis (CFPS) and affinity purification and enzymatic cleavage scheme could overcome the challenges of delivering sensitive biologic medicines. This platform was successfully used to synthesize peptide hormones which are used to treat various diseases. With further development, it has the potential to revolutionize the decentralized manufacturing of peptide hormone drugs.
ACS SYNTHETIC BIOLOGY
(2023)