Progress in molecular biology and translational science最新文献

{"title":"Cell-free systems for expression of proteins and enzymes.","authors":"Eeba, Jhalak Aggarwal, Nisheeth Agarwal","doi":"10.1016/bs.pmbts.2025.08.002","DOIUrl":"https://doi.org/10.1016/bs.pmbts.2025.08.002","url":null,"abstract":"<p><p>Once restricted to cellular boundaries, cell-free expression has advanced to the forefront of contemporary biotechnology, heralding a novel paradigm in protein and enzyme biosynthesis. Cell-free expression system (CFES) offers an exquisite platform for production of these biomolecules in an open and controlled environment, liberated from the constraints of cellular regulations. The CFES facilitates precise manipulation of expression dynamics, thereby enabling the synthesis of proteins that are otherwise challenging to express in conventional hosts. Initially conceived as a pivotal tool for deciphering the genetic code, this technology has evolved into a sophisticated and versatile system for biosynthesis of proteins and enzymes, empowering researchers with unprecedented control over the molecular machinery of life. Biosynthesis of toxic proteins and complex enzymes in a flexible and highly regulated cell-free environment offers a panoply of applications in diagnostics, therapeutics, biomanufacturing and synthetic biology. From rudimentary extract-based systems to highly advanced and well-developed systems such as protein synthesis using recombinant elements (PURE), cell-free expression has profoundly reshaped the scientific landscape in ways that were previously unimaginable. Since the time of their inception, CFESs have elegantly exemplified the fusion of engineering and biological insights, thereby advancing the field from a technical curiosity to a treasure trove of possibilities. This chapter traces the journey of cell-free expression of proteins and enzymes followed by an extensive review of different types and formats of CFESs. Finally, the chapter ends by providing brief description of various applications, challenges and revolutionary promises that the cell-free systems behold for future.</p>","PeriodicalId":21157,"journal":{"name":"Progress in molecular biology and translational science","volume":"219 ","pages":"21-49"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146195539","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cell-free systems for production of therapeutics.","authors":"Sandeep Kaur, Gholamreza Abdi, Maryam Abbasi Tarighat, Vaseem Raja, Mudassir Ahmad Bhat","doi":"10.1016/bs.pmbts.2025.10.004","DOIUrl":"https://doi.org/10.1016/bs.pmbts.2025.10.004","url":null,"abstract":"<p><p>The development of the cell-free system (CFS) is transforming the manufacturing landscape of biomolecules with therapeutic value by providing a flexible and convenient alternative to cell-based expression systems. Compared to cell-based systems, cell-free systems offer advantages such as better modularity, greater control, and faster production because they utilize the basic transcription and translation machinery of various organisms, including Escherichia coli, wheat germ, and mammalian cells. The performance and applicability of these platforms have significantly improved in recent years through advances in synthetic biology, metabolic engineering, lyophilized formulations, and system optimization. These developments have made them more suitable for high-throughput screening and rapid prototyping, as well as on-demand biomanufacturing, especially in low-resource or emergency settings. This chapter explores the concepts, technology, engineering, and therapeutic applications of CFS. It critically examines issues related to scalability, cost-efficiency, and post-translational modifications, along with the future prospects of the field. The emerging needs combining rapid, on-demand, and personalized medicine with modern healthcare, pandemic preparedness, and decentralized bioproduction highlight cell-free systems as a vital part of the future of therapeutic development and delivery.</p>","PeriodicalId":21157,"journal":{"name":"Progress in molecular biology and translational science","volume":"219 ","pages":"51-92"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146195597","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cell free systems for biodesign.","authors":"Mohd Tariq, Nil Patil, Mukul Jain, Dhruv Desai, Piyusha Kuhite, Ayush Madan, Sandeep Rawat","doi":"10.1016/bs.pmbts.2025.08.010","DOIUrl":"https://doi.org/10.1016/bs.pmbts.2025.08.010","url":null,"abstract":"<p><p>Cell-free systems (CFS) have emerged as transformative tools in synthetic biology, enabling the execution of complex biological reactions such as transcription and translation outside the confines of living cells. By eliminating the cellular membrane, CFS allows unprecedented control over biochemical conditions, facilitating rapid prototyping (up to 10x faster than traditional in vivo systems), streamlined design-build-test cycles, and the direct production of proteins, including those that are toxic or difficult to express in vivo. Rooted in pivotal discoveries from the 1960s, CFS technologies have evolved to include refined systems like the PURE system, freeze-dried diagnostics, and programmable biosynthesis platforms, integrating seamlessly with automation, artificial intelligence, and microfluidics. Modern CFS platforms support a broad range of applications, from on-demand vaccine and therapeutic production to environmental monitoring, protein engineering, and sustainable biomanufacturing. Their modular nature makes them ideal for developing genetic circuits, metabolic pathways, and biosensors, while also accelerating high-throughput screening and educational access through platforms like BioBits. Despite challenges such as reagent costs, batch variability, and scalability, recent advances in energy regeneration, lyophilization, and predictive modelling are progressively addressing these hurdles. Ultimately, CFS is not just a powerful research tool; it represents a paradigm shift toward decentralized, programmable biotechnology. From field-deployable diagnostics to space-based biomolecule synthesis, cell-free systems are paving the way for a more responsive, accessible, and innovative future in biological engineering.</p>","PeriodicalId":21157,"journal":{"name":"Progress in molecular biology and translational science","volume":"218 ","pages":"219-248"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146047247","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cell-free systems for expression of transmembrane protein.","authors":"Kinza Farooq, Syed Damin Abbas Hamdani, Mustafeez Mujtaba Babar, Jayakumar Rajadas","doi":"10.1016/bs.pmbts.2025.09.001","DOIUrl":"https://doi.org/10.1016/bs.pmbts.2025.09.001","url":null,"abstract":"<p><p>Transmembrane proteins (TMPs) are the most important components of cellular membranes They mediate a wide array of biological processes, including signal transduction, ion and metabolite transport, enzymatic catalysis, and cell-cell communication. Due to their central roles in physiological homeostasis and pathophysiological disorders, TMPs are considered prime targets in drug discovery, accounting for more than half of all therapeutic targets. Besides their essential activity in human proteome, their characterization faces several challenges in expression, folding and stability in conventional cell-based systems. Cell-free protein synthesis (CFPS) is a good alternative which offers an open, reproducible, and high-yield platform for TMP production that bypasses many limitations associated with in vivo expression. Fundamental principles, advancements, and applications of CFPS in the context of TMP expression are discussed in this chapter. It highlights various CFPS platforms both from prokaryotic and eukaryotic sources and covers their respective strengths in the production of TMPs. The discussion covers biochemical approaches, such as the incorporation of membrane mimetics, folding chaperones, and non-standard amino acids, as well as technological innovations including hybrid systems, microfluidic reactors, and high-throughput automation. Achieving correct topology and functional reconstitution are thoroughly examined, along with future directions involving modular membrane systems and next-generation synthetic biology tools. By boosting the accessibility and precision of TMP production, CFPS has potential to redefine structural biology, drug discovery, and the development of novel therapeutics. By enabling precise, rapid, and scalable production of TMPs, CFPS has the potential to redefine workflows in structural biology, drug screening, biosensor development, and the production of next-generation biologics.</p>","PeriodicalId":21157,"journal":{"name":"Progress in molecular biology and translational science","volume":"219 ","pages":"1-19"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146195534","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cell-free protein synthesis systems for post-translational modifications.","authors":"Khushal Khambhati, Khushbu Panchal, Suresh Ramakrishna, Vijai Singh","doi":"10.1016/bs.pmbts.2025.10.001","DOIUrl":"https://doi.org/10.1016/bs.pmbts.2025.10.001","url":null,"abstract":"<p><p>Post-translational modification (PTM) is an important event of the cell. It aids function and behavior to the proteins. It is highly regulated and reported to govern various phenomenon of cells. Moreover, dysfunctionality in PTM also drives diseases development. Approximately 650 types of PTM have been reported so far, of which phosphorylation, methylation, acetylation, ubiquitination, glycosylation, lipidation, citrullination, and SUMOylation are among the most studied till date. Many pharmaceutical valued proteins are also post-translationally modified. In order to investigate or use such polypeptide as therapeutics, homogeneous population of targeted protein with authentic PTM is required. Conventional in vivo approaches pose challenges to produce a population of proteins with correct PTM. However, with the gain of interest to examine the PTM among the researchers across the globe, cell-free protein synthesis (CFPS) platforms emerged as a tool of choice to quench valuable insights. As found in vivo, CFPS mediated PTM of proteins enables the user to acquire the structural and functional properties of protein with ease. Several strategies such as GlycoPRIME for glycosylation, soluble expression strategies such as SIMPLEx, orthogonal translation systems for the site specific phosphoserine insertion, continuous exchange CFPS system for hydroxylation, PURE based systems for acetylation, and cell engineering for robust CFPS mediated disulfide bond formation has been proposed over the years. Current chapter describes such CFPS mediated strategy to produce proteins with PTM. Overall, CFPS mediated approaches provide a controlled and efficient means to produce proteins with defined PTMs, facilitating mechanistic studies and therapeutic applications.</p>","PeriodicalId":21157,"journal":{"name":"Progress in molecular biology and translational science","volume":"219 ","pages":"171-187"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146195593","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cell-free systems for vaccine production.","authors":"Nhat Le Bui, Khanh Linh Nguyen, Bao Phan Van, Yen Nhi Khuong, Dinh-Toi Chu","doi":"10.1016/bs.pmbts.2025.08.001","DOIUrl":"https://doi.org/10.1016/bs.pmbts.2025.08.001","url":null,"abstract":"<p><p>Cell-free (CF) systems is harness cellular components including tRNAs, ribosomes, and polymerase to synthesize proteins in vitro. Owing to their significant CF systems offer substantial advantages over traditional cell-based systems, including higher speed, biosafety, and portability. As a result, CF systems have emerged as a powerful platform for biomedical research, with particularly promising applications in biosensing and diagnostics, protein production, synthetic biology and vaccine development. In this chapter, we provided a comprehensive overview of CF system applications in the field of biomedical sciences, with an emphasis on vaccine development and production. We also discussed their successful applications in the expression of antigens from challenging pathogens, such as Plasmodium falciparum, Chlamydia muridarum, and SARS-CoV-2. Moreover, this chapter proposed several promising innovations to address current limitations of CF platforms such as the shortage of post-translational modifications, endotoxin presence, and high production cost. Emerging solutions include glycoengineering to introduce functional glycosylation, freeze-drying for improving storage and distribution, exosome-based delivery for designing next generation vaccines, and even machine learning integration, to optimize the production pipelines.</p>","PeriodicalId":21157,"journal":{"name":"Progress in molecular biology and translational science","volume":"219 ","pages":"93-106"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146195589","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cell-free systems for nanobiomaterials assembly.","authors":"Anjana Eettikkal Mathew, Vishnu Kirthi Arivarasan","doi":"10.1016/bs.pmbts.2025.11.002","DOIUrl":"https://doi.org/10.1016/bs.pmbts.2025.11.002","url":null,"abstract":"<p><p>Nanobiomaterials-engineered constructs operating at the 1-100 nm scale-integrate biological macromolecules such as nucleic acids and proteins with synthetic polymers or inorganic nanocomponents to achieve programmable functionality in biomedical and industrial contexts. Representative systems, including DNA origami-based drug carriers, CRISPR-Cas delivery scaffolds, and artificial protein vesicles, demonstrate subcellular targeting precision exceeding 90 % and up to a fivefold enhancement in intratumoral drug accumulation relative to conventional nanocarriers. Despite these advances, in vivo nanomanufacturing remains constrained by cytotoxicity, intracellular metabolic load, and limited spatiotemporal control over synthetic parameters. Cell-free systems (CFS) mitigate these limitations by utilizing crude lysates from prokaryotic or eukaryotic cells that retain functional transcription-translation machinery while eliminating the constraints of cellular viability. Platforms such as the reconstituted PURE system enable high-throughput, template-directed synthesis of nanoscale architectures incorporating noncanonical elements, including synthetic polymers, fluorinated analogs, and unnatural amino acids. This open, tunable environment permits over fivefold increases in yields of cytotoxic or aggregation-prone peptides, accelerates DNA origami prototype fabrication to under 24 h, and supports assembly of hybrid enzyme-polymer conjugates with retained catalytic activity. Distinct CFS sources impart complementary advantages: bacterial extracts offer rapid, cost-effective protein expression; yeast lysates facilitate eukaryote-specific glycosylation for ligand-specific nanocapsules; and mammalian systems enable near-physiological post-translational modification essential for therapeutic nanobiomaterials. Coupling CFS with artificial intelligence-based design optimization and microfluidic automation now underpins a new paradigm of programmable, scalable nanobiomanufacturing. By decoupling molecular construction from living systems, cell-free biofabrication establishes a controllable, high-fidelity platform for the rational engineering of nano-bio hybrid systems in precision medicine, biosensing, and tissue regeneration.</p>","PeriodicalId":21157,"journal":{"name":"Progress in molecular biology and translational science","volume":"219 ","pages":"189-210"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146195582","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cell-free systems for development of biosensors.","authors":"Stuti Ganatra, Alok Pandya","doi":"10.1016/bs.pmbts.2025.09.003","DOIUrl":"https://doi.org/10.1016/bs.pmbts.2025.09.003","url":null,"abstract":"<p><p>Cell-free systems (CFSs) have become powerful tools in synthetic biology, enabling the creation of fast, modular, and customizable biosensors without relying on living cells. By utilizing in vitro transcription and translation, these systems offer a finely controlled biochemical environment suitable for sensing applications in fields such as healthcare, environmental science, agriculture, and food quality assurance. This chapter provides an in-depth look at the design and functionality of CFS-based biosensors, highlighting the construction of genetic circuits, signal output strategies, and device formats including paper-based platforms, microfluidic systems, and wearable technologies. With use cases ranging from pathogen detection to monitoring environmental contaminants, cell-free biosensors are proving especially valuable in point-of-care (POC) and low-resource settings. The chapter also addresses current limitations such as shelf-life, sensitivity, and scalability and explores engineering solutions including AI-assisted design, molecular optimization, and advanced material integration. Looking ahead, the convergence of CFS biosensing with smart technologies such as IoT and distributed fabrication promises a new era of accessible, intelligent diagnostics.</p>","PeriodicalId":21157,"journal":{"name":"Progress in molecular biology and translational science","volume":"218 ","pages":"129-156"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146047204","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-throughput screening of biomolecules using cell-free systems.","authors":"Brahmjot Singh, Jyoti, Suhail Kapta, Sandeep Kaur, Ajay Kumar, Gholamreza Abdi","doi":"10.1016/bs.pmbts.2025.11.001","DOIUrl":"https://doi.org/10.1016/bs.pmbts.2025.11.001","url":null,"abstract":"<p><p>High-throughput screening (HTS) has revolutionized the identification and evaluation of biomolecules by enabling the parallel testing of large libraries of compounds, nucleic acids, and proteins against biological targets. Traditionally conducted in live cells, HTS faces limitations such as cellular toxicity, metabolic interference, and regulatory constraints. Cell-free systems (CFS), which operate in vitro using reconstituted transcription-translation machinery, have emerged as powerful alternatives. These systems circumvent the constraints of cellular physiology, allowing for rapid and tunable expression of biomolecules directly from DNA or RNA templates. This chapter explores the principles, platforms, and applications of CFS-based HTS, highlighting its transformative impact on synthetic biology, drug discovery, diagnostics, and protein engineering. Several cell-free systems are detailed, including those derived from E. coli, wheat germ, rabbit reticulocytes, and the defined PURE system. The integration of CFS with high-throughput platforms such as microplates, droplet microfluidics, and paper-based devices enables cost-effective, scalable, and multiplexed assays. Analytical readouts, including fluorescence, luminescence, mass spectrometry, and digital PCR, provide real-time, sensitive detection of biochemical outputs. Furthermore, automation and machine learning are increasingly incorporated through robotic liquid handling and data-driven DBTL cycles, accelerating discovery and design processes. Despite challenges such as high reagent costs and limited post-translational modifications, innovations such as lyophilized CFS kits, artificial cells, and AI-integrated closed-loop platforms are expanding the frontiers of HTS. Altogether, CFS-based HTS offers a flexible, rapid, and accessible approach for next-generation biomolecular screening and therapeutic development.</p>","PeriodicalId":21157,"journal":{"name":"Progress in molecular biology and translational science","volume":"218 ","pages":"187-217"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146047250","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"New frontiers and applications of cell-free systems.","authors":"Khushbu Panchal, Khushal Khambhati, Viswanathaiah Matam, Suresh Ramakrishna, Vijai Singh","doi":"10.1016/bs.pmbts.2025.10.002","DOIUrl":"https://doi.org/10.1016/bs.pmbts.2025.10.002","url":null,"abstract":"<p><p>Cell-free systems (CFS) have emerged as a key platform in the field of synthetic biology. This is used to understand natural biological systems outside living cells. It contains cell extracts from procaryotes, eucaryotes, which provides a controlled environment for complex biological processes that leads to the synthesis of valuable biomolecules. It lacks natural mechanisms, yet it contains all the necessary components, which are required for the synthesis of desired biomolecules. It is specifically designed for the elimination of barriers to molecular transport across cell membranes. This chapter highlights a basic CFS and its various applications, such as high-throughput protein synthesis and expression, non-canonical amino acids incorporation in proteins, biosensors, drug discovery and in the metabolic engineering. This chapter also focuses on the various case studies and recent advancements to study how these systems are used for the transformation of biotechnology and provides rapid, more adaptable, and affordable solutions in the field of research as well as industrial levels. Altogether, CFS emerged as promising platform in the field of biotechnology, biomedicine, and environmental sustainability.</p>","PeriodicalId":21157,"journal":{"name":"Progress in molecular biology and translational science","volume":"218 ","pages":"1-17"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146047231","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}