A cell-free workflow for detecting and characterizing RiPP recognition element-precursor peptide interactions DOI Creative Commons
Derek A. Wong, Zachary M. Shaver, Maria D. Cabezas

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Март 26, 2024

Abstract Post-translational modifications (PTMs) are important for the stability and function of many therapeutic proteins peptides. Current methods studying engineering PTM installing often suffer from low-throughput experimental techniques. Here we describe a generalizable, in vitro workflow coupling cell-free protein synthesis (CFPS) with AlphaLISA rapid expression testing proteins. We apply our to two representative classes peptide therapeutics: ribosomally synthesized post-translationally modified peptides (RiPPs) conjugate vaccines. First, demonstrate how can be used characterize binding activity RiPP recognition elements, an first step biosynthesis, integrated into biodiscovery pipeline computationally predicted products. Then, adapt study engineer oligosaccharyltransferases (OSTs) involved vaccine production, enabling identification mutant OSTs sites within carrier that enable high efficiency production In total, expect will accelerate design-build-test cycles PTMs.

Язык: Английский

Targeting Enterotoxins: Advancing Vaccine Development for Enterotoxigenic Escherichia coli ETEC DOI Creative Commons

Josune Salvador-Erro,

Yadira Pastor, Carlos Gamazo

и другие.

Toxins, Год журнала: 2025, Номер 17(2), С. 71 - 71

Опубликована: Фев. 6, 2025

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal disease worldwide, particularly in children low- and middle-income countries. Its ability to rapidly colonize the intestinal tract through diverse colonization factors toxins underpins its significant public health impact. Despite extensive research several vaccine candidates reaching clinical trials, no licensed exists for ETEC. This review explores temporal spatial coordination ETEC virulence factors, focusing on interplay between adherence mechanisms toxin production as critical targets therapeutic intervention. Advancements molecular biology host–pathogen interaction studies have uncovered species-specific variations cross-reactivity human animal strains. In particular, heat-labile (LT) heat-stable (ST) provided crucial insights into disruption. Additional exotoxins, such EAST-1 hemolysins, further highlight multifactorial nature pathogenicity. Innovative strategies, including multiepitope fusion antigens (MEFAs), mRNA-based approaches, glycoconjugates, aim enhance broad-spectrum immunity. Novel delivery methods, like intradermal immunization, show promise eliciting robust immune responses. Successful vaccination against will offer an effective affordable solution with potential greatly reduce mortality prevent stunting, representing highly impactful cost-efficient global challenge.

Язык: Английский

Процитировано

1
Glycoconjugate vaccines against antimicrobial resistant pathogens DOI Creative Commons
Charlotte Sorieul, Marta Dolce, Maria Rosaria Romano

и другие.

Expert Review of Vaccines, Год журнала: 2023, Номер 22(1), С. 1055 - 1078

Опубликована: Окт. 30, 2023

Introduction Antimicrobial resistance (AMR) is responsible for the death of millions worldwide and stands as a major threat to our healthcare systems, which are heavily reliant on antibiotics fight bacterial infections. The development vaccines against main pathogens involved urgently required prevention remains essential rise AMR.

Язык: Английский

Процитировано

17
Immunogenicity of Current and Next-Generation Pneumococcal Conjugate Vaccines in Children: Current Challenges and Upcoming Opportunities DOI Creative Commons
Kristen A. Feemster, Ulrike K. Buchwald,

Natalie Banniettis

и другие.

Open Forum Infectious Diseases, Год журнала: 2024, Номер 11(5)

Опубликована: Апрель 26, 2024

Abstract Global use of pneumococcal conjugate vaccines (PCVs) with increasingly broader serotype coverage has helped to reduce the burden disease in children and adults. In clinical studies comparing PCVs, higher-valency PCVs have met noninferiority criteria (based on immunoglobulin G geometric mean concentrations response rates) for most shared serotypes. A numeric trend declining immunogenicity against serotypes also been observed; however, relevance is uncertain, warranting additional research evaluate effectiveness new vaccines. Novel conjugation processes, carriers, adjuvants, vaccine platforms are approaches that could help maintain or improve subsequent while achieving protection increasing valency

Язык: Английский

Процитировано

6
Construction of an Escherichia coli chassis for efficient biosynthesis of human-like N-linked glycoproteins DOI Creative Commons

Zixin Bao,

Yuting Gao, Yitong Song

и другие.

Frontiers in Bioengineering and Biotechnology, Год журнала: 2024, Номер 12

Опубликована: Март 20, 2024

The production of N -linked glycoproteins in genetically engineered Escherichia coli holds significant potential for reducing costs, streamlining bioprocesses, and enhancing customization. However, the construction a stable low-cost microbial cell factory efficient humanized -glycosylated recombinant proteins remains formidable challenge. In this study, we developed glyco-engineered E. chassis to produce with human-like glycan Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc-, containing human glycoform Gal-β-1,4-GlcNAc-β-1,3-. Our initial efforts were replace various loci genome XL1-Blue strain oligosaccharyltransferase PglB glycosyltransferases LsgCDEF construct chassis. addition, systematically optimized promoter regions regulate transcription levels. Subsequently, utilizing plasmid carrying target protein, have successfully obtained 100% tetrasaccharide modification at yield approximately 320 mg/L. Furthermore, constructed metabolic pathway sialylation using dual-expression cassette protein CMP-sialic acid synthesis cell, resulting 40% efficiency terminal α-2,3- 65 mg/L homogeneously sialylated flasks. findings pave way further exploration producing different linkages (α-2,3/α-2,6/α-2,8) -glycoproteins periplasm plug-and-play chassis, laying strong foundation industrial-scale production.

Язык: Английский

Процитировано

4
Chemical biology tools to probe bacterial glycans DOI Creative Commons

Daniel Calles-Garcia,

Danielle H. Dube

Current Opinion in Chemical Biology, Год журнала: 2024, Номер 80, С. 102453 - 102453

Опубликована: Апрель 5, 2024

Bacterial cells are covered by a complex carbohydrate coat of armor that allows bacteria to thrive in range environments. As testament the importance bacterial glycans, effective and heavily utilized antibiotics including penicillin vancomycin target disrupt glycocalyx. Despite their importance, study glycans lags far behind eukaryotic counterparts. use large palette monosaccharides craft leading molecules significantly more than refractory study. Fortunately, chemical tools designed probe have yielded insights into these molecules, structures, biosynthesis, functions.

Язык: Английский

Процитировано

4
Cell-Free Gene Expression: Methods and Applications DOI Creative Commons
Andrew C. Hunt, Blake J. Rasor, Kosuke Seki

и другие.

Chemical Reviews, Год журнала: 2024, Номер 125(1), С. 91 - 149

Опубликована: Дек. 19, 2024

Cell-free gene expression (CFE) systems empower synthetic biologists to build biological molecules and processes outside of living intact cells. The foundational principle is that precise, complex biomolecular transformations can be conducted in purified enzyme or crude cell lysate systems. This concept circumvents mechanisms have evolved facilitate species survival, bypasses limitations on molecular transport across the wall, provides a significant departure from traditional, cell-based rely microscopic cellular "reactors." In addition, cell-free are inherently distributable through freeze-drying, which allows simple distribution before rehydration at point-of-use. Furthermore, as nonliving, they provide built-in safeguards for biocontainment without constraints attendant genetically modified organisms. These features led increase development use CFE over past two decades. Here, we discuss recent advances highlight how transforming efforts cells, control genetic networks, manufacture biobased products.

Язык: Английский

Процитировано

4
Plant-made trained immunity-based vaccines: Beyond one approach DOI
Elizabeth Monreal‐Escalante, Miriam Angulo, Abel Ramos‐Vega

и другие.

International Journal of Pharmaceutics, Год журнала: 2025, Номер unknown, С. 125572 - 125572

Опубликована: Апрель 1, 2025

Язык: Английский

Процитировано

0
Preparation and Evaluation of Novel Epitope-Based ETEC K88-K99 Bivalent Vaccine DOI Creative Commons
Shuangshuang Wang, Yuxin Yang,

Xinru Yue

и другие.

Veterinary Sciences, Год журнала: 2025, Номер 12(4), С. 381 - 381

Опубликована: Апрель 18, 2025

Enterotoxigenic Escherichia coli (ETEC) is one of the primary pathogens causing diarrhea in piglets, significant economic losses swine farming industry. Due to numerous serotypes ETEC, traditional vaccines fail provide sufficient cross-protection, and subunit based on epitope design have emerged as a safer more effective approach for prevention control. Unlike vaccine development strategies that involve tandem arrangement multiple antigenic epitopes, this study used K88-FaeG protein backbone incorporated epitopes K99-FanC achieve better immunogenicity. By using bioinformatics software predict B-cell linear (score over 0.6), from three-dimensional structures (50% amino acid score ≥0.2), IgG antibody subtypes, well docking analysis with Sus scrofa aminopeptidase N (APN) receptors, six were selected. Through Western blotting competitive ELISA, we confirmed all recombinant proteins exhibited binding capabilities K88- K99-positive serum. The ELISA results showed serum levels specific IgA antibodies increased after immunization, FaeG-Ep3 FaeG-Ep5 inducing highest titers against FanC-IgG (Log2 = 14.96) FaeG-IgG 17.96), respectively. Bacterial adhesion assays revealed only effectively blocked both K99 K88 IPEC-J2 cells. Immunization challenge experiments that, unimmunized group, mice infected experienced weight loss (p < 0.05) intestinal villus shedding wall structural damage. However, FaeG-Ep3-immunized no occurred infection, protection rate (83%) was same FaeG FanC immunized groups. Overall, identified shows potential application value provides new insights developing multivalent ETEC.

Язык: Английский

Процитировано

0
Scalable Cell‐Free Production of Active T7 RNA Polymerase DOI

R. Rezvani,

Rochelle Aw, J. Chan

и другие.

Biotechnology and Bioengineering, Год журнала: 2025, Номер unknown

Опубликована: Апрель 29, 2025

ABSTRACT The SARS‐CoV‐2 pandemic highlighted the urgent need for biomanufacturing paradigms that are robust and fast. Here, we demonstrate rapid process development scalable cell‐free production of T7 RNA polymerase, a critical component in mRNA vaccine synthesis. We carry out 1‐L gene expression (CFE) reaction achieves over 90% purity, low endotoxin levels, enhanced activity relative to commercial polymerase. To achieve this demonstration, implement rolling circle amplification circumvent difficulties DNA template generation, tune conditions, such as temperature, additives, purification tags, agitation, boost yields. similar quality titer polymerase more than four orders magnitude volume. This proof principle positions CFE viable solution decentralized biotherapeutic manufacturing, enhancing preparedness future public health crises or emergent threats.

Язык: Английский

Процитировано

0
GlycoCAP: A Cell-Free, Bacterial Glycosylation Platform for Building Clickable Azido-Sialoglycoproteins DOI
Ariel Helms Thames, Sam J. Moons, Derek A. Wong

и другие.

ACS Synthetic Biology, Год журнала: 2023, Номер 12(4), С. 1264 - 1274

Опубликована: Апрель 11, 2023

Glycan-binding receptors known as lectins represent a class of potential therapeutic targets. Yet, the targeting remains largely untapped due in part to limitations tools for building glycan-based drugs. One group desirable structures is proteins with noncanonical glycans. Cell-free protein synthesis systems have matured promising approach making glycoproteins that may overcome current and enable new glycoprotein medicines. this has not been applied construction To address limitation, we develop cell-free platform glycans and, specifically, clickable azido-sialoglycoproteins (called GlycoCAP). The GlycoCAP uses an Escherichia coli-based system site-specific installation onto high degree homogeneity efficiency. As model, construct four dust mite allergen (Der p 2): α2,3 C5-azido-sialyllactose, C9-azido-sialyllactose, α2,6 C9-azido-sialyllactose. Through series optimizations, achieve more than 60% sialylation efficiency azido-sialic acid. We then show azide click handle can be conjugated model fluorophore using both strain-promoted copper-catalyzed chemistry. anticipate will facilitate development discovery drugs by granting access wider variety possible glycan also provide functionalizing chemistry conjugation.

Язык: Английский

Процитировано

8