Biotechnology Research Research Articles

Photoinduced Transformations with Diverse Maleimide Scaffolds

Maleimides serve as crucial components in various synthetic processes and are of significant interest to researchers in bioorganic chemistry and biotechnology. Although thermal reactions involving maleimides have been studied extensively, light-mediated reactions with maleimides remain relatively underutilized. This review focuses on understanding the behavior of maleimides in their excited state, particularly their role as synthetic scaffolds for excited-state reactions. Specific emphasis is placed on the diverse photoreactions involving maleimides and photophysical evaluation from our research group.

Biosensor applications in the monitoring of elderly patients

Nurse-based patient monitoring is prone to errors due to manual measurements and documentation, leading to potential inaccuracies in care. The use of biosensors offers a promising solution by enabling real-time and continuous monitoring of patient health. Categorizing patient care reports as critical or non-critical using mobile recording systems based on biosensor data can help prevent errors and improve care. The use of biosensors can significantly reduce morbidity and mortality, especially after emergencies and accidents. These devices improve the quality of care and increase the satisfaction of older people, their families and healthcare professionals. Wearable biosensors make it easier for older people to monitor their health, which can help reduce hospital admissions. Chronic diseases such as cardiovascular disease, cancer, diabetes, dementia and stroke pose challenges to healthcare delivery and interpretation of results. Integrating biosensors into health monitoring and measurement is an innovative approach to managing these chronic conditions more effectively. To improve self-management of chronic diseases in older people, it is essential to educate healthcare professionals and promote research in this area. As a result, the use of biosensors to monitor the daily activities and health parameters of elderly patients is expanding, highlighting the importance of multidisciplinary research in biotechnology, chemistry, engineering and nursing.

The Assembly of Miniaturized Droplets toward Functional Architectures.

Recent explorations of bioengineering have generated new concepts and strategies for the processing of soft and functional materials. Droplet assembly techniques can address problems in the construction of extremely soft architectures by expanding the manufacturing capabilities using droplets containing liquid or hydrogels including weak hydrogels. This Perspective sets out to provide a brief overview of this growing field, and discusses the challenges and opportunities ahead. The study highlights the recent key advances of materials and architectures from hitherto effective droplet-assembly technologies, as well as the applications in biomedical and bioengineering fields from artificial tissues to bioreactors. It is envisaged that these assembled architectures, as nature-inspired models, will stimulate the discovery of biomaterials and miniaturized platforms for interdisciplinary research in health, biotechnology, and sustainability.

Generating and characterizing a comprehensive panel of CHO cells glycosylation mutants for advancing glycobiology and biotechnology research

This report describes the development and characterization of a comprehensive collection of CHO cell glycosylation mutants with significant potential for advancing glycobiology and biotechnology. EPO-Fc and trastuzumab, two model molecules, were produced using these mutants to assess the effects of mutated glycogenes, and LC-MS/MS analysis was employed to quantitatively analyse their N-glycans. EPO-Fc exhibited exclusively homogeneous Man9 glycans only when nearly all α-mannosidases in the genome were inactivated, except lysosomal MAN2B1. Some mutants lacking GnT-I activity produce mostly Man5 N-glycans, while their O-glycan and glycolipid profiles can differ due to other mutations in the cell. GnT-II deficiency prevents GnT-V from adding GlcNAc to the core N-glycan, resulting in branches attaching solely to the α1,3-linked mannose, leaving the α1,6-linked mannose free. The mutant-produced antibody’s single-branched glycan contains more sialic acid than the dual-branched glycans produced in CHO-K1 cells. Trastuzumab produced in these mutants provided insights into how Fc N-glycans impact the antibody’s interaction with FcγR1 and FcγR2a, FcγR3a, and their influence on antibody-dependent cellular cytotoxicity (ADCC). In the study of Fc glycans in Fc-FcγR1 and FcγR2a interactions, we observed a consistent glycan-related impact on binding to both receptors, indicating a common interaction mechanism between Fc glycans and both FcγRI and FcγRIIa. CHO mutants produced trimeric gp120 demonstrated distinct reactivity with multiple broadly neutralizing anti-HIV antibodies, confirming the involvement of gp120 glycans in interactions with specific broadly neutralizing antibodies. Finally, one of the mutants produced human β-glucocerebrosidase with uniform Man5 N-glycans, showcasing its potential for glycoengineered production and enhancement in therapeutic efficacy.

Structural study of the intrinsically disordered tardigrade damage suppressor protein (Dsup) and its complex with DNA

Studies of proteins, found in one of the most stress-resistant animals tardigrade Ramazzottius varieornatus, aim to reveal molecular principles of extreme tolerance to various types of stress and developing applications based on them for medicine, biotechnology, pharmacy, and space research. Tardigrade DNA/RNA-binding damage suppressor protein (Dsup) reduces DNA damage caused by reactive oxygen spices (ROS) produced upon irradiation and oxidative stresses in Dsup-expressing transgenic organisms. This work is focused on the determination of structural features of Dsup protein and Dsup-DNA complex, which refines details of protective mechanism. For the first time, intrinsically disordered nature of Dsup protein with highly flexible structure was experimentally proven and characterized by the combination of small angle X-ray scattering (SAXS) technique, circular dichroism spectroscopy, and computational methods. Low resolution models of Dsup protein and an ensemble of conformations were presented. In addition, we have shown that Dsup forms fuzzy complex with DNA.

Diversity of Mycotoxin-Producing Fungi in Leafy and Fruit Vegetables Sold in Port Harcourt Metropolis, Nigeria

Mycotoxins are secondary metabolites produced by fungi and are capable of causing diseases and even death in both humans and other animals. This study investigated the diversity of mycotoxin-producing fungi from leafy (Telfairia occidentalis and Bassica oleracea) and fruit (Solanum lycopersicum and Cucumis sativus) vegetables sold in Port Harcourt metropolis, Nigeria using molecular techniques. The fungi were isolated from vegetable samples showing signs of disease using standard methods. DNA extraction and Polymerase chain reaction were carried out at the molecular laboratory of Regional Centre of Biotechnology and Bioresources research, University of Port Harcourt. The isolates were screened for the presence of mycotoxigenic genes (nor1 (aflatoxin), tri6 (trichothecene), otanps (Ochratoxin A), fum13 (fumonisin) and ZEA (zearalenone)) using PCR technique. The isolates containing mycotoxins were identified using ITS gene sequences. Ten out of the eighteen isolates were positive for four (nor1, tri6, otanps and fum13) out of the five mycotoxigenic genes screened. The nine isolates included fungi belong to six genera: Meyerozyma, Pithomyces, Fusarium, Trametes, Penicillium and Aspergillus. The ten isolates were classified as Meyerozyma carbbica RCBBR_Sf13, Pithomyces chartarum RCBBR_Sf5, Fusarium falciforme RCBBR_Sf10, Trametes duplexa RCBBR_Sf17, Trametes versicolor RCBBR_Sf2a, Trametes duplexa RCBBR_Mf1, Fusarium longifundum RCBBR_Mf4, Penicillium soosanum RCBBR_Mf7, Aspergillus aflatoxiformans RCBBR_Sf9, Fusarium circinatum RCBBR_Sf3 based on their ITS gene sequences. Their ITS gene sequences have been deposited in GenBank under the accession numbers OR816039-OR816047, with the exception of Fusarium circinatum RCBBR_Sf3 which could not accession. This study has demonstrated that mycotoxin-producing fungi are diverse and widespread in leafy and fruit vegetables sold within Port Harcourt Metropolis. This raises both public health and food security concerns.

Characterization of genetic diversity in Eragrostis tef (zucc.) Trotter through microsatellite and morphological markers

Eragrostis tef (Zucc.) Trotter is Ethiopia's most significantly important grain crop; however, its productivity is quite low (1.7 tons per hectare) due to a variety of issues, including the poor yield potential of varieties produced thus far. To achieve future production improvements in tef, comprehensive study of the crop's genetic variability and variety, utilizing both genetic and morphological markers, necessary. The study was designed to assess the genetic diversity of 64 tef genotypes utilizing 10 Simple Sequence Repeats (SSRs) markers. This investigation took place at the Agricultural Biotechnology Research Center in Holeta, Ethiopia. Field phenotypic diversity evaluation was conducted at two distinct locations: Debre Zeit and Worabe, applied a simple lattice design during 2020/21 season. Molecular analysis of variance demonstrated a substantial proportion of variation among individuals (46 %), followed closely variation among individuals (43 %), with the least variation observed within population (11 %). Concurrently, analysis of morphological data, encompassing twenty phenotypic characteristics, revealed significant variation (P ≤ 0.01) among almost all the tested genotypes for recorded parameters, as indicated by combined analysis of variance across sites. These findings underscore the high diversity among the studied genotypes, suggesting a considerable potential for crop improvement through direct selection and intra-specific hybridization strategies. From the perspectives of both conservation and utilization of tef genetic materials, there is a pressing need for more extensive and systematic molecular level research. Promoting field trials and genotype-by-environment interaction research is crucial. These efforts will enhance our understanding of tef genetic and contribute to its effective conservation and utilization.

Determinants of Food Security in Tobacco and Sugarcane Production Zones, Migori County, Kenya

At 34%, food insecurity in Migori County is considered alarming while its measurement has posed challenges to academicians and researchers whilst many studies exist on food security determinants, none has been done in Migori entailing tobacco producers, sugarcane growers and non-growers in Kuria and Migori sub-counties. The study examined food security determinants guided by a consumer theory and measured by means of HFIAS modelled in ologit model. Data collection was done using an open data kit pre-loaded with a questionnaire and analyzed using stata software wherein results depicted 38.89% as severely insecure, 33.33% moderately insecure, 9.26% mildly insecure and 18.52% food secure while household size, off-farm income, capital sourcing was significant at p<0.001 in varying degrees and categories after ologit regression analysis. There is need for the national government to provide cash crop insurance and fixed contracts to prevent risks associated with price fluctuations as well as invest in innovative biotechnology research through formulation of policies to aid in ensuring capacity building through its stakeholders such as the national research institutes and other donor agencies.

Relics of interspecific hybridization retained in the genome of a drought-adapted peanut cultivar.

Peanut (Arachis hypogaea L.) is a globally important oil and food crop frequently grown in arid, semi-arid, or dryland environments. Improving drought tolerance is a key goal for peanut crop improvement efforts. Here we present the genome assembly and gene model annotation for 'Line8', a peanut genotype bred from drought tolerant cultivars. Our assembly and annotation are the most contiguous and complete peanut genome resources currently available. The high contiguity of the Line8 assembly allowed us to explore structural variation both between peanut genotypes and subgenomes. We detect several large inversions between Line8 and other peanut genome assemblies, and there is a trend for the inversions between more genetically diverged genotypes to have higher gene content. We also relate patterns of subgenome exchange to structural variation between Line8 homeologous chromosomes. Unexpectedly, we discover that Line8 harbors an introgression from A.cardenasii, a diploid peanut relative and important donor of disease resistance alleles to peanut breeding populations. The fully resolved sequences of both haplotypes in this introgression provide the first in situ characterization of A.cardenasii candidate alleles that can be leveraged for future targeted improvement efforts. The completeness of our genome will support peanut biotechnology and broader research into the evolution of hybridization and polyploidy.

Three-Dimensionally Printed Microsystems to Facilitate Flow-Based Study of Cells from Neurovascular Barriers of the Retina.

Rapid prototyping has produced accessible manufacturing methods that offer faster and more cost-effective ways to develop microscale systems for cellular testing. Commercial 3D printers are now increasingly adapted for soft lithography, where elastomers are used in tandem with 3D-printed substrates to produce in vitro cell assays. Newfound abilities to prototype cellular systems have begun to expand fundamental bioengineering research in the visual system to complement tissue engineering studies reliant upon complex microtechnology. This project used 3D printing to develop elastomeric devices that examined the responses of retinal cells to flow. Our experiments fabricated molds for elastomers using metal milling, resin stereolithography, and fused deposition modeling via plastic 3D printing. The systems were connected to flow pumps to simulate different flow conditions and examined phenotypic responses of endothelial and neural cells significant to neurovascular barriers of the retina. The results indicated that microdevices produced using 3D-printed methods demonstrated differences in cell survival and morphology in response to external flow that are significant to barrier tissue function. Modern 3D printing technology shows great potential for the rapid production and testing of retinal cell responses that will contribute to both our understanding of fundamental cell response and the development of new therapies. Future studies will incorporate varied flow stimuli as well as different extracellular matrices and expanded subsets of retinal cells.

Characterization of UV-absorbing Mycosporine-like Amino Acids in Freshwater Cyanobacterium Nostoc calcicola

Mycosporine-like amino acids (MAAs) are gaining attention for their UV photoprotective properties and potential applications in biomedical fields, cosmetics, and toiletries. In this study, Nostoc calcicola a genus of cyanobacteria distributed in freshwater were tested for MAA production. This organism was provided by the Freshwater Algae Culture Collection of the Institute of Hydrobiology, the Chinese Academy of Sciences. It was isolated and cultured in the laboratory, in 500 ml glass flasks containing 300 ml of liquid medium BG11 treated on UV-B light 0.1 Wm–2. At different time, cultures were exposed to UV-B light at intensity 0.1 W. m-1 to determine the Maximum PSII photochemical (Fv/Fm). MAAs were extracted using 100% HPLC grade methanol and separated through high-performance liquid chromatography and liquid chromatography/mass spectrometry. Based on liquid chromatography/mass spectrometry analysis, one MAA compound was identified as porphyra. Thus, UV-B treatment of Nostoc calcicola significantly enhanced the production of UV-absorbing mycosporine-like amino acids (MAAs). The content of porphyra could reach up to 8.5 mg/g dry weight in Nostoc calcicola under UV-B treatments for 2 days. The present cyanobacteria could be used for biotechnology research and the UV-absorbing compound porphyra may be of great value in the development of novel sunscreens.

A Review of the Regulatory Challenges of Personalized Medicine.

Personalized medicine integrates genomics with clinical and familial histories, revolutionizing healthcare by tailoring treatments to individual patient characteristics. At its core, pharmacogenomics enables the customization of medication prescriptions based on genetic profiles, enhancing drug efficacy and safety. This precision medicine approach addresses disease diagnosis, prevention, and treatment, offering targeted therapies for conditions like autoimmune disorders, rheumatoid arthritis, and neoplastic conditions. Examples of pharmacogenomics and personalized medicine include treatment for certain conditions like blood clotting disorders (warfarin (blood thinner), genetic variability, acute lymphoblastic leukemia (ALL), and thiopurine methyltransferase (TPMT) testing) in leukemia treatment. Historically, personalized medicine has evolved from Hippocrates' humoral theories to modern molecular diagnostics. The shift from cellular to molecular-level investigations has led to the current post-genomic era, emphasizing the four chemical components of DNA in understanding and treating disorders. This evolution enhances our ability to predict disease susceptibility, treatment response, and potential adverse reactions, demanding advancements in privacy laws, payment systems, regulatory standards, and education. Personalized healthcare optimizes treatment by considering genetic, environmental, and lifestyle factors, reducing adverse reactions, and improving patient satisfaction. It drives genomic and biotechnological research, fostering the development of targeted therapies and diagnostic tools, and streamlining drug development. Applications in lung cancer, renal carcinoma, and rheumatoid arthritis (RA) illustrate the efficacy of personalized medicine. Targeted therapies, such as tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs), show significant success in lung cancer. Biomarkers guide renal carcinoma treatments, while gene expression profiles predict rheumatoid arthritis outcomes with tumor necrosis factor-alpha (TNF-α) blockers. The US FDA's increasing approvals of personalized treatments underscore its commitment to precision medicine. However, regulatory challenges persist, necessitating clear processes and policies. Ethical considerations, including equitable access and privacy, are critical for responsible implementation. The FDA's increasing approvals of personalized treatments highlight its commitment to advancing precision medicine, yet regulatory challenges remain, requiring the development of clear processes and policies to manage innovation safely. While these hurdles are being addressed through evolving guidelines and collaborative efforts, ethical considerations, including equitable access and privacy, are critical for responsible implementation. Early examples, such as warfarin and imatinib, demonstrate the role of genetic information in guiding treatment, illustrating the potential of personalized medicine to optimize healthcare outcomes.

Nanotechnology Advances in the Detection and Treatment of Lymphoid Malignancies.

Lymphoid malignancies are complex diseases with distinct biological behaviors, clinical presentations, and treatment responses. Ongoing research and advancements in biotechnology enhance the understanding and management of these malignancies, moving towards more personalized approaches for diagnosis and treatment. Nanotechnology has emerged as a promising tool to improve some limitations of conventional diagnostics as well as treatment strategies for lymphoid malignancies. Nanoparticles (NPs) offer unique advantages such as enhanced multimodal detection, drug delivery, and targeted therapy capabilities, with the potential to improve precision medicine and patient outcomes. Here, we comprehensively examine the current landscape of nanoconstructs applied in the management of lymphoid disease. Through a comprehensive analysis of preclinical studies, we highlight the translational potential of NPs in revolutionizing the field of hematological malignancies, with a specific focus on lymphoid neoplasms.

Impact of NaCl stress on phytoconstituents and bioactivity of Matricaria chamomilla: a multi-analytical approach

Matricaria chamomilla (Asteraceae), commonly known as chamomile can tolerate freezing temperatures and grows in many soil types. This plant is found on all continents and has significant medicinal value. There are more than 120 chemicals detected in chamomile flowers, with the majority found in the essential oil. In this study, M. chamomilla was given the NaCl stress of 0 mM, 1 mM, 100 mM, and 150 mM concentrations This study was the first to assess the efficacy of German chamomile upon exposure to salt stress hence plant particles that had been dried and powdered were analyzed using, phytochemical tests, Fourier Transform Infrared and UV–Vis spectroscopy, thin layer chromatography, fluorescence recovery after photobleaching assay, antibacterial and antioxidant activity. The characterization and results of these activities show amazing results which enhance their antibacterial property with an increased zone of inhibition when the samples of salt stress of the above-given concentrations were compared to the control samples. More graph analysis indicates an effective impact of salt stress on the phytoconstituents of M. chamomilla. Other than that, there was a clear flower induction upon salt stress, as a variety of compounds are regarded as essential to the biological functions of chamomile flowers according to the phytoconstituent screening which can be further used in the cosmetic industry, pharmaceutical industry, and all other fields as well for various application as a nano-drug or bio-drug. Due to this, this plant became essential for plant biotechnology research.

Three-way junction-mediated three-letter coded SDA cascade CRISPR/Cas12a system for circRNA detection

Circular RNAs (circRNAs), a special type of non-coding RNAs with covalently closed circular structure, have emerged as promising liquid biopsy biomarker. However, the precise detection of circRNAs is severely hampered by interference from homologous linear RNAs, posing a significant obstacle for their applications in molecular diagnostics. Herein, a strategy named three-way junction (TWJ)-mediated three-letter coded strand displacement amplification (SDA) cascade CRISPR/Cas12a system (TTSC) has been devised to achieve precise detection of circRNAs. Three-letter coded SDA (TC-SDA) has been originally designed in this study for eliminating undesired interference from linear RNA and performing dual functionality in both discrimination and amplification. With this design, the proposed strategy elegantly distinguishes between circRNA and homologous linear RNA through proximity effect of TWJ structure and non-specific amplification blocking ability of the three-letter code. Ultimately, this strategy successfully identified circRNA down to 0.1 % abundance. In addition, benefiting from the synergistically accelerated multiple signal amplification in one step, TTSC strategy exhibited good linear relationship of 0.5–1000 pM with a detection limit of 80.6 fM. Notably, the recovery test indicated that the proposed strategy has good prospects for clinical applications. Therefore, TTSC is expected to provide an ideal platform for circRNA detection in biotechnology research and molecular diagnostics.

A fast and genotype-independent in planta Agrobacterium-mediated transformation method for soybean

Efficient genotype-independent transformation and genome editing are highly desirable for plant biotechnology research and product development efforts. We have developed a novel approach to enable fast, high-throughput, and genotype-flexible Agrobacterium-mediated transformation using the important crop soybean as a test system. This new method is called GiFT (genotype-independent fast transformation) and involves only a few simple steps. The method uses germinated seeds as explants, and DNA delivery is achieved through Agrobacterium infection of wounded explants as in conventional in vitro-based methods. Following infection, the wounded explants are incubated in liquid medium with a sublethal level of selection and then transplanted directly into soil. The transplanted seedlings are then selected with herbicide spray for 3 weeks. The time required from initiation to fully established healthy T0 transgenic events is about 35 days. The GiFT method requires minimal in vitro manipulation or use of tissue culture media. Because the regeneration occurs in planta, the GiFT method is highly flexible with respect to genotype, which we demonstrate via successful transformation of elite germplasms from diverse genetic backgrounds. We also show that the soybean GiFT method can be applied to both conventional binary vectors and CRISPR-Cas12a vectors for genome editing applications. Analyses of T1 progeny demonstrate that the events have a high inheritance rate and can be used for genome engineering applications. By minimizing the need for tissue culture, the novel approach described here significantly improves operational efficiency while greatly reducing personnel and supply costs. It is the first industry-scale transformation method to utilize in planta selection in a major field crop.

A multiplexed sensing system with multimodal readout electronics and thread-based electrochemical sensors for high throughput screening

High throughput screening (HTS) based on microwell (or bioreactor) arrays is a well-established approach for rapid screening and identification of lead molecules or target analytes for drug discovery and other applications in biotechnology. However, it has been challenging to scale up these platforms due to the lack of integrated sensors to monitor the microenvironment of each microwell in the array needed. In this work, we present a practical solution for a miniaturized multiplexed sensing system with integrated circuitry capable of performing continuous electrochemical potentiometric and amperometric measurements. The platform uses lightweight multiplexed electrochemical sensors implemented on thin textile threads, all of which share a single operational amplifier (op-amp) and a switch network for both voltage and current measurements. The resulting platform forgoes the need for large-area sensors or bulky multichannel potentiostat with a simple readout circuitry capable of multimodal measurements. The design and validation of the multiplexed sensing system have been reported in the context of measuring pH and oxygen (O2), which are crucial biomarkers for any HTS application. The thread-based pH and O2 sensors possess sensitivities of ∼65.87 mV/pH for the pH sensor (n = 3) and 0.6621 nA/mg L-1 for the O2 sensor (n = 3). This architecture is modular such that it can be easily extended to monitoring multiple biomarkers employing thread-based sensors and sharing the same readout circuitry, making it a practical and versatile tool for biotechnology research.

AI-Assisted Rational Design and Activity Prediction of Biological Elements for Optimizing Transcription-Factor-Based Biosensors.

The rational design, activity prediction, and adaptive application of biological elements (bio-elements) are crucial research fields in synthetic biology. Currently, a major challenge in the field is efficiently designing desired bio-elements and accurately predicting their activity using vast datasets. The advancement of artificial intelligence (AI) technology has enabled machine learning and deep learning algorithms to excel in uncovering patterns in bio-element data and predicting their performance. This review explores the application of AI algorithms in the rational design of bio-elements, activity prediction, and the regulation of transcription-factor-based biosensor response performance using AI-designed elements. We discuss the advantages, adaptability, and biological challenges addressed by the AI algorithms in various applications, highlighting their powerful potential in analyzing biological data. Furthermore, we propose innovative solutions to the challenges faced by AI algorithms in the field and suggest future research directions. By consolidating current research and demonstrating the practical applications and future potential of AI in synthetic biology, this review provides valuable insights for advancing both academic research and practical applications in biotechnology.

Improvement and application of vacuum-infiltration system in tomato.

The Agrobacterium-mediated transient expression system has been developed and applied to various plants as an alternative to stable transformation. However, its application in tomatoes is still limited due to low expression efficiency. In this study, we describe an improved vacuum-infiltration system that can be used in both tomato fruits and leaves. Notably, this study is the first report of vacuum infiltration in attached tomato fruits. The feasibility of the improved vacuum-infiltration system in Micro-Tom tomato was confirmed by various assays, including multiple fluorescent protein expression analysis, β-glucuronidase activity analysis, and RUBY reporter visualization. Subsequently, the improved vacuum-infiltration system was successfully applied to tomato biotechnology research. Herein, a trichome-specific promoter in tomato was identified that can drive the directional synthesis of specific plant natural products (PNPs). Additionally, based on the assessment results of the improved vacuum-infiltration system, we obtained a flavonoid-rich tomato variety through the stable transformation of AmRosea and AmDelila. In a significant practical application, we successfully synthesized the high-value scutellarin in tomato, which provides an alternative route for the production of PNPs from plants. In addition, the improved vacuum-infiltration system has been demonstrated to be suitable for commercial tomato varieties ('Emerald' and 'Provence') as well. The improved vacuum-infiltration system not only speeds up fundamental and applied research in tomato but also offers an additional powerful tool for advancing tomato synthetic biology research.

On-Demand Thio-Succinimide Hydrolysis for the Assembly of Stable Protein-Protein Conjugates.

Chemical post-translational protein-protein conjugation is an important technique with growing applications in biotechnology and pharmaceutical research. Maleimides represent one of the most widely employed bioconjugation reagents. However, challenges associated with the instability of first- and second-generation maleimide technologies are yet to be fully addressed. We report the development of a novel class of maleimide reagents that can undergo on-demand ring-opening hydrolysis of the resulting thio-succinimide. This strategy enables rapid post-translational assembly of protein-protein conjugates. Thio-succinimide hydrolysis, triggered upon application of chemical, photochemical, or enzymatic stimuli, allowed homobifunctional bis-maleimide reagents to be applied in the production of stable protein-protein conjugates, with complete temporal control. Bivalent and bispecific protein-protein dimers constructed from small binders targeting antigens of oncological importance, PD-L1 and HER2, were generated with high purity, stability, and improved functionality compared to monomeric building blocks. The modularity of the approach was demonstrated through elaboration of the linker moiety through a bioorthogonal propargyl handle to produce protein-protein-fluorophore conjugates. Furthermore, extending the functionality of the homobifunctional reagents by temporarily masking reactive thiols included in the linker allowed the assembly of higher order trimeric and tetrameric single-domain antibody conjugates. The potential for the approach to be extended to proteins of greater biochemical complexity was demonstrated in the production of immunoglobulin single-domain antibody conjugates. On-demand control of thio-succinimide hydrolysis combined with the facile assembly of chemically defined homo- and heterodimers constitutes an important expansion of the chemical methods available for generating stable protein-protein conjugates.