Globular Proteins Research Articles

Deciphering Membrane Proteins Through Deep Learning Models by Revealing Their Locale Within the Cell

Membrane proteins constitute essential biomolecules attached to or integrated into cellular and organelle membranes, playing diverse roles in cellular processes. Their precise localization is crucial for understanding their functions. Existing protein subcellular localization predictors are predominantly trained on globular proteins; their performance diminishes for membrane proteins, explicitly via deep learning models. To address this challenge, the proposed study segregates membrane proteins into three distinct locations, including the plasma membrane, internal membrane, and membrane of the organelle, using deep learning algorithms including recurrent neural networks (RNN) and Long Short-Term Memory (LSTM). A redundancy-curtailed dataset of 3000 proteins from the MemLoci approach is selected for the investigation, along with incorporating pseudo amino acid composition (PseAAC). PseAAC is an exemplary technique for extracting protein information hidden in the amino acid sequences. After extensive testing, the results show that the accuracy for LSTM and RNN is 83.4% and 80.5%, respectively. The results show that the LSTM model outperforms the RNN and is most commonly employed in proteomics.

Deep-GB: A novel deep learning model for globular protein prediction using CNN-BiLSTM architecture and enhanced PSSM with trisection strategy.

Globular proteins (GPs) play vital roles in a wide range of biological processes, encompassing enzymatic catalysis and immune responses. Enzymes, among these globular proteins, facilitate biochemical reactions, while others, such as haemoglobin, contribute to essential physiological functions such as oxygen transport. Given the importance of these considerations, accurately identifying Globular proteins is essential. To address the need for precise GP identification, this research introduces an innovative approach that employs a hybrid-based deep learning model called Deep-GP. We generated two datasets based on primary sequences and developed a novel feature descriptor called, Consensus Sequence-based Trisection-Position Specific Scoring Matrix (CST-PSSM). The model training phase involved the application of deep learning techniques, including the bidirectional long short-term memory network (BiLSTM), gated recurrent unit (GRU), and convolutional neural network (CNN). The BiLSTM and CNN were hybridised for ensemble learning. The CST-PSSM-based ensemble model achieved the most accurate predictive outcomes, outperforming other competitive predictors across both training and testing datasets. This demonstrates the potential of harnessing deep learning for precise GB prediction as a robust tool to expedite research, streamline drug discovery, and unveil novel therapeutic targets.

Genetically Engineered Liposwitch-Based Nanomaterials.

Fusion of intrinsically disordered and globular proteins is a powerful strategy to create functional nanomaterials. However, the immutable nature of genetic encoding restricts the dynamic adaptability of nanostructures postexpression. To address this, we envisioned using a myristoyl switch, a protein that combines allostery and post-translational modifications─two strategies that modify protein properties without altering their sequence─to regulate intrinsically disordered protein (IDP)-driven nanoassembly. A typical myristoyl switch, allosterically activated by a stimulus, reveals a sequestered lipid for membrane association. We hypothesize that this conditional exposure of lipids can regulate the assembly of fusion proteins, a concept we term "liposwitching". We tested this by fusing recoverin, a calcium-dependent myristoyl switch, with elastin-like polypeptide, a thermoresponsive model IDP. Biophysical analyses confirmed recoverin's myristoyl-switch functionality, while dynamic light scattering and cryo-transmission electron microscopy showed distinct calcium- and lipidation-dependent phase separation and assembly. This study highlights liposwitching as a viable strategy for controlling DP-driven nanoassembly, enabling applications in synthetic biology and cellular engineering.

Identification and characterization of the lipoprotein N-acyltransferase in Bacteroides

Members of the Bacteroidota compose a large portion of the human gut microbiota, contributing to overall gut health via the degradation of various polysaccharides. This process is facilitated by lipoproteins, globular proteins anchored to the cell surface by a lipidated N-terminal cysteine. Despite their importance, lipoprotein synthesis by these bacteria is understudied. In Escherichia coli, the α-amino-linked lipid of lipoproteins is added by the lipoprotein N-acyltransferase Lnt. Herein, we have identified a protein distinct from Lnt responsible for the same process in Bacteroides, named lipoprotein N-acyltransferase in Bacteroides (Lnb). Deletion of Lnb yields cells that synthesize diacylated lipoproteins, with impacts on cell viability and morphology, growth on polysaccharides, and protein composition of membranes and outer membrane vesicles (OMVs). Our results not only challenge the accepted paradigms of lipoprotein biosynthesis in gram-negative bacteria but also suggest the existence of a new family of lipoprotein N-acyltransferases.

Silicon-Containing Dendrimer with Long Spacers between Branching Points

Dendrimers are macromolecules with a highly ordered, hyperbranched globular structure, which is ideal for a number of high-tech applications owing to the monodispersity, well-defined functionality, and combinatorial potential. In this communication, the synthesis of the first-generation silicon-containing dendrimer with long spacers between branching points using a combination of polyaddition reactions is presented.

Optimization of extraction for efficient recovery of kenaf seed protein isolates: evaluation of physicochemical and techno-functional characteristics.

Kenaf seeds are a rich source of protein; however, finding the best extraction method is crucial to obtaining high-quality protein from these underutilized seeds. This research devised an optimized extraction process for best recovery of kenaf seeds protein using response surface methodology. The key parameters affecting the yield and protein content were optimized, including extraction pH (2-11), seed:water ratio (5:1-50:1), temperature (30-90 °C), and duration (20-360 min). The physicochemical and techno-functional properties of kenaf seed protein isolates (KSPIs) were examined. A maximum protein yield of 12.05 g/100 g with purity level 91.94 g/100 g was obtained using an optimized extraction with pH 11.0, seed:water ratio 50:1, 360 min duration, and temperature 50 °C. The oil and water retention capacities of KSPI were 1.14 mL g-1 and 1.37 mL g-1 respectively. After 30 min at pH 7, KSPIs demonstrated remarkable emulsion capacity (83.12%) and stability (75.63%), along with high foaming capacity (106%) and stability (18.3%). As per high-performance liquid chromatography analysis, arginine, glutamic acid, leucine, phenylalanine, and lysine were the most abundant amino acids detected in KPSIs. The KSPIs' globular protein structure was successfully verified using analytical approaches, including Fourier transform infrared spectroscopy, protein fraction ratios, and differential scanning calorimetry. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that KPSI has a molecular weight distribution ranging from 10 kDa to 50 kDa. The results of this study support the application of the proposed response-surface-methodology-optimized extraction method for efficient recovery of high-quality kenaf seed proteins that meet the necessary physicochemical and techno-functional requirements. © 2024 Society of Chemical Industry.

Characterization of rapeseed protein supramolecular structures obtained by aqueous extractions

Aqueous extraction of rapeseed has the potential to produce mildly refined yet functional protein ingredients. However, the colloidal structures of these mildly refined ingredients are still not fully understood. Therefore, the aim of this work was to examine the structures present in rapeseed extracts by analyzing their scattering characteristics across different length scales. Rapeseed protein concentrates were obtained after aqueous extractions at acidic (pH 5.7) or alkaline (pH 8.5) conditions. The structures were investigated using complementary techniques, cryo-transmission electron microscopy, small angle X-ray scattering and small angle neutron scattering. To understand the scattering contribution of various components at characteristic length scales, D2O contrast variation was performed, and SAXS analysis for extracts at pH 5.7 treated with pectinase were compared to those untreated. All suspensions contained globular proteins within a network of soluble polysaccharides as observed by Cryo-TEM. SANS analysis with different D2O/H2O ratios demonstrated that at low q, both proteins and polysaccharide contributed to scattering, suggesting the presence of large complexes containing both. The signal for pH 8.5 suspensions was mainly attributed to oleosomes. SAXS measurements agreed with the SANS data, and pH 5.7 suspensions measured after pectin hydrolysis revealed that the complexes were formed by rapeseed globulins (∼4 nm) and polysaccharide chains. This research highlights the importance of understanding the protein-polysaccharide interactions occurring in rapeseed aqueous extracts, to fully utilize these less refined fractions as value-added ingredients in future sustainable foods.

Quantitative characterization of non-specific interaction of two globular proteins with Dextran T70 in a binary mixture.

In a bid to quantify the contribution of molecular structure to non-specific interactions leading to functionally important structural changes in cellular processes, the self-interaction of dextran-T70 (DT70) and its interaction with each of bovine serum albumin (BSA) and ovomucoid trypsin inhibitor (OVO) were studied at pH 7.4 between 5 and 37°C. The dependences of the apparent molecular weight of each of BSA, OVO and DT70 on the concentration of DT70 were independent of temperature. The activity coefficient of the interaction of each species on DT70 concentration was also independent of temperature. The change in activity coefficient was however dependent on the molecular structure and size of the interacting species. The energy of insertion of each macromolecule in DT70 increased in the order DT70 > BSA > OVO. These findings show that although the enthalpic contribution is negligible, the extent of the entropic contribution to the macromolecular activity coefficient of interaction is chiefly the consequence of the exclusion volume of the interacting macromolecules.

The Potential Application of Mung Bean (Vigna radiata L.) Protein in Plant‐Based Food Analogs: A Review

ABSTRACTThe increasing demand for plant‐based food products by consumers along with the growing global population requires the discovery of novel and sustainable protein sources to address environmental challenges and meet nutritional requirements. Among various plant proteins, mung bean protein (MBP) exhibits several unique characteristics that make it a valuable ingredient in the field of plant‐based food analogs. This literature review aims to express the unique structure and composition of MBP, in addition to its physicochemical, functional, and nutritional properties. Furthermore, its potential applications in novel meat, dairy, and egg analogs are highlighted to meet the growing demand for sustainable, nutritious, and delicious plant‐based food alternatives. Structurally, MBP consists of a complex arrangement of amino acids, forming a globular protein with distinct functional properties. Its composition is also rich in essential amino acids, particularly leucine and lysine, making it a promising protein source for plant‐based diets. From a techno‐functional perspective, MBP exhibits remarkable gelling, emulsifying, foaming, and binding properties, which are necessary for the development of stable emulsions, airy foams, firm gels, and cohesive textures in various plant‐based food formulations. Moreover, MBP and its derivatives can possess notable bio‐functional properties, including antioxidant activity and anti‐inflammatory benefits, as well as cholesterol‐lowering, anti‐obesity, antidiabetic, antihypertensive, and anticancer effects. Consequently, the production of food analogs based on MBP not only improves the techno‐functional attributes of the final products but also can promote consumer health and well‐being.

Initiation of ERAD by the bifunctional complex of Mnl1 mannosidase and protein disulfide isomerase.

Misfolded glycoproteins in the endoplasmic reticulum (ER) lumen are translocated into the cytosol and degraded by the proteasome, a conserved process called ER-associated protein degradation (ERAD). In S. cerevisiae , the glycan of these proteins is trimmed by the luminal mannosidase Mnl1 (Htm1) to generate a signal that triggers degradation. Curiously, Mnl1 is permanently associated with protein disulfide isomerase (Pdi1). Here, we have used cryo- electron microscopy, biochemical, and in vivo experiments to clarify how this complex initiates ERAD. The Mnl1-Pdi1 complex first de-mannosylates misfolded, globular proteins that are recognized through a C-terminal domain (CTD) of Mnl1; Pdi1 causes the CTD to ignore completely unfolded polypeptides. The disulfides of these globular proteins are then reduced by the Pdi1 component of the complex, generating unfolded polypeptides that can be translocated across the membrane. Mnl1 blocks the canonical oxidative function of Pdi1, but allows it to function as the elusive disulfide reductase in ERAD.

Ligand presentation controls collective MSC response to matrix stress relaxation in hybrid PEG-HA hydrogels

Cell interactions with the extracellular matrix (ECM) influence intracellular signaling pathways related to proliferation, differentiation, and secretion, amongst other functions. Herein, bone-marrow derived mesenchymal stromal cells (MSCs) are encapsulated in a hydrazone crosslinked hyaluronic acid (HA) hydrogel, and the extent of stress relaxation is controlled by systemic introduction of irreversible triazole crosslinks. MSCs form elongated multicellular structures within hydrogels containing RGD peptide and formulated with elastic composition slightly higher than the hydrogel percolation threshold (12 % triazole, 88 % hydrazone). A scaling analysis is presented (<RgStructure2>12 ∼Nα) to quantify cell-material interactions within these structures with the scaling exponent (α) describing either elongated (0.66) or globular (0.33) structures. Cellular interactions with the material were controlled through peptides to present integrin binding ECM cues (RGD) or cadherin binding cell-cell cues (HAVDI) and MSCs were observed to form highly elongated structures in RGD containing hydrogels (α=0.56±0.05), whereases collapsed structures were observed within HAVDI containing hydrogels (α=0.39±0.04). Finally, cytokine secretion was investigated, and a global increase in secreted cytokines was observed for collapsed structures compared to elongated. Taken together, this study presents a novel method to characterize cellular interactions within a stress relaxing hydrogel where altered cluster morphology imparts changes to cluster secretory profiles.

Drop-Cast Hybrid Poly(styrene)-b-Poly(ethylene oxide) Metal Salt Films: Solvent Evaporation and Crystallinity-Dependent Evolution of Film Morphology.

Morphology templates of solution-based diblock copolymer (DBC) films with loading metal salts are widely applied in photocatalysts, photovoltaics, and sensors due to their adjustable characteristics based on surface (de-)wetting and microphase separation. The present work investigates the morphologies of drop-cast hybrid films based on poly(styrene)-b-poly(ethylene oxide) (PS-b-PEO) and the metal salts titanium isopropoxide (TTIP) and zinc acetate dehydrate (ZAD) in comparison to the pure DBC. By utilizing scanning electron microscopy, grazing-incidence small- and wide-angle X-ray scattering, and differential scanning calorimetry, we find that the resulting film morphologies depend not only on the presence of metal salts but also on solvent evaporation and crystalline formation. At 20 °C, additional TTIP and ZAD in the polymer template cause the morphology to change from packed globular structures to separated wormlike structures attributed to the changed polymer environment. Furthermore, additional tetrahydrofuran causes irregular structures at the precursor film part and the overlapped wormlike structures to transition into close-packed globular structures at the cap film parts of the pure DBC. In contrast, at 50 °C, the globular structures transit to fingerprint patterns due to the thermal behavior of the crystallizable PEO blocks, and the metal salt additives suppress crystalline structure formation in the PEO domains.

Templating Iron(III) Oxides on DNA Molecules.

Fe(III) oxides were prepared as free nanoparticles and on DNA templates via the precipitation of Fe(III) salts with NaOH in the presence/absence of DNA. Through control of the pH and temperature, FeOOH and Fe2O3 were synthesised. The formation of templated materials FeOOH/DNA and Fe2O3/DNA was confirmed using UV-Vis absorption and FTIR spectra. The direct optical gap of Fe2O3/DNA was estimated as 3.2 eV; the absorption by FeOOH/DNA and Fe2O3/DNA at longer wavelengths is weaker, but consistent with indirect gaps near 2 eV. X-ray photoelectron spectra confirmed the presence of Fe(III) and DNA in the templated samples. Analysis of the X-ray diffraction patterns of both templated and non-templated FeOOH and Fe2O3 demonstrated that the materials were the α-FeOOH and α-Fe2O3 polymorphs with crystallite diameters of the DNA-templated materials estimated as 7.6 nm and 6.8 nm. Transmission electron microscopy showed needle-like crystals of both FeOOH and Fe2O3, but the Fe2O3 contains some globular structures. In contrast, the morphology of FeOOH/DNA and Fe2O3/DNA consists of needle-like crystallites of the respective oxides organised into complex dendritic structures with a length on the 10 μm scale formed by the DNA molecules. Finally, scanned conductance microscopy provided evidence for the conductivity of the FeOOH/DNA after alignment via molecular combing on an Si/SiO2 substrate. Fe2O3/DNA did not exhibit any detectable conductivity.

Globular proteins as Pickering emulsion stabilizers: Particles or surfactants?

Can globular proteins be considered as Pickering emulsion stabilizers? In recent years, there has been a growing interest in the study of Pickering emulsions due to their unique properties. Among these, protein-stabilized Pickering emulsions have garnered significant attention, thanks to their environmentally friendly nature and wide-ranging applications across various industries. Pickering emulsions are typically classified separately from surfactant-stabilized emulsions due to their distinct stabilization mechanisms and remarkable resistance to coalescence. Although proteins have long been considered as particles in the context of Pickering emulsions, a debate still exists regarding their classification. In this work, we aim to address this debate by investigating the rheological and interfacial activity aspects of bovine serum albumine (BSA) proteins at silicone oil-water interfaces in the context of silicone oil-in-water emulsions. For rheological analysis and comparisons, emulsions were also formulated with surfactants and particles. Our results show that proteins can present both surfactant-like and particle-like behavior in terms of interfacial activity and microscopic structures with varying formulation parameters (pH). Rheological modelling, additionally, indicate similar behavior between emulsions with particles, surfactants and proteins. Thus, the challenge of categorizing proteins definitively as one or the other persists.

Thermo- and Photoresponsive Smart Nanomaterial Based on Poly(diethyl vinyl phosphonate)-Capped Gold Nanoparticles.

A new nanodevice based on gold nanoparticles (AuNPs) capped with poly(diethylvinylphosphonate) (PDEVP) has been synthesized, showing interesting photophysical and thermoresponsive properties. The synthesis involves a properly designed Yttriocene catalyst coordinating the vinyl-lutidine (VL) initiator active in diethyl vinyl phosphonate polymerization. The unsaturated PDEVP chain ending was thioacetylated, deacetylated, and reacted with tetrachloroauric acid and sodium borohydride to form PDEVP-VL-capped AuNPs. The NMR, UV-Vis, and ESI-MS characterization of the metal nanoparticles confirmed the formation of the synthetic intermediates and the expected colloidal systems. AuNPs of subnanometric size were determined by WAXD and UV-Vis analysis. UV-Vis and fluorescence analysis confirmed the effective anchoring of the thiolated PDEVP to AuNPs. The formation of 50-200 nm globular structures was assessed by SEM and AFM microscopy in solid state and confirmed by DLS in aqueous dispersion. Hydrodynamic radius studies showed colloidal contraction with temperature, demonstrating thermoresponsive behavior. These properties suggest potential biomedical applications for the photoablation of malignant cells or controlled drug delivery induced by light or heat for the novel PDEVP-capped AuNP systems.

Functional Diversification of Sec13 Isoforms for Storage Protein Trafficking in Rice Endosperm Cells.

Coat protein complex II (COPII) vesicles play crucial roles in mediating the endoplasmic reticulum (ER) exit of newly synthesized proteins to the Golgi in eukaryotic cells. However, the molecular functions of COPII components and their functional diversifications in plant seeds remain obscure. Here, we showed that the rice (Oryza sativa) glutelin precursor accumulation12 (gpa12) mutant is defective in storage protein export from the ER, resulting in the formation of aggregated protein bodies. Map-based cloning revealed that GPA12 encodes a COPII outer layer protein, Sec13a, that mainly localizes to endoplasmic reticulum exit sites (ERES) and partially localizes to the Golgi. Biochemical experiments verified that Sec13a physically interacts with Sec31 and Sec16, and mutation in Sec13 compromises its interaction with Sec31 and Sec16, thereby affecting the membrane association of the inner complex components Sar1b and Sec23c. Apart from Sec13a, the rice genome encodes two other Sec13 isoforms, Sec13b and Sec13c. Notably, we observed an abnormal accumulation of globular ER structures in the sec13bc double mutant but not in the single mutants, suggesting a functional redundancy of Sec13b and Sec13c in modulating ER morphology. Taken together, our results substantiated that Sec13a plays an important role in regulating storage protein export from the ER, while Sec13b and Sec13c are required for maintaining ER morphology in rice endosperm cells. Our findings provide insights into the functional diversification of COPII components in plants.

Realizing active targeting in cancer nanomedicine with ultrasmall nanoparticles.

Ultrasmall nanoparticles (usNPs) have emerged as promising theranostic tools in cancer nanomedicine. With sizes comparable to globular proteins, usNPs exhibit unique physicochemical properties and physiological behavior distinct from larger particles, including lack of protein corona formation, efficient renal clearance, and reduced recognition and sequestration by the reticuloendothelial system. In cancer treatment, usNPs demonstrate favorable tumor penetration and intratumoral diffusion. Active targeting strategies, incorporating ligands for specific tumor receptor binding, serve to further enhance usNP tumor selectivity and therapeutic performance. Numerous preclinical studies have already demonstrated the potential of actively targeted usNPs, revealing increased tumor accumulation and retention compared to non-targeted counterparts. In this review, we explore actively targeted inorganic usNPs, highlighting their biological properties and behavior, along with applications in both preclinical and clinical settings.

Cooling Rate and Compositional Effects on Microstructural Evolution and Mechanical Properties of (CoCrCuTi)100-xFex High-Entropy Alloys.

This study investigates the impact of cooling rate and alloy composition on phase formations and properties of (CoCrCuTi)100-xFex (x = 0, 5, 10, 12.5, 15) high-entropy alloys (HEAs). Samples were synthesized using arc-melting and electromagnetic levitation, followed by quenching through the use of a Cu chill or V-shaped Cu mold. Cooling rates were evaluated by measuring dendrite arm spacings (DASs), employing the relation DAS = k ɛ-n, where constants k = 16 and n = ½. Without Fe addition, a microstructure consisting of BCC1 + BCC2 phases formed, along with an interdendritic (ID) FCC Cu-rich phase. However, with the addition of 5-10% Fe, a Cu-lean C14 Laves phase emerged, accompanied by a Cu-rich ID FCC phase. For cooling rates below 75 K/s, alloys containing 10% Fe exhibited liquid phase separation (LPS), characterized by globular Cu-rich structures within the Cu-lean liquid. In contrast, for the same composition, higher cooling rates of 400-700 K/s promoted a dendritic/interdendritic microstructure. Alloys with 12.5-15 at. % Fe displayed LPS irrespective of the cooling rate, although an increase in uniformity was noted at rates exceeding 700 K/s. Vickers hardness and fracture toughness generally increased with Fe content, with hardness ranging from 444 to 891 HV. The highest fracture toughness (5.5 ± 0.4 KIC) and hardness (891 ± 66 HV) were achieved in samples containing 15 at. % Fe, cooled at rates of 25-75 K/s.

On the abundance and importance of AXXXA sequence motifs in globular proteins and their involvement in Cβ[sbnd]Cβ interaction

The AXXXA and GXXXG motifs are frequently observed in helices, especially in membrane proteins. The motif GXXXG is known to stabilize helix-helix association in membrane proteins via CαHO bonding. AXXXA sequence motif additionally stabilizes the folded state of proteins. We found 27,000 and 18,000 occurrences of AXXXA and GXXXG motifs in a non-redundant set of 6000 obligate homodimeric (OD) complexes. Interestingly, this is less pronounced in transient homodimers (TD) and heterodimers (HetD). On average each obligate homodimer contains four AXXXA motifs, it is 2 and 3.5 for HetD and TD, respectively. Focusing on the binding surface it is seen that 27 % of the ODs contain at least one AXXXA motif at the interface, whereas it is 17 % and 15 % for HetD and TD respectively. AXXXA predominantly stabilizes the OD quaternary structure via the side chain CβCβ interactions. This interaction is energetically favorable and is found to be a major driving force for OD quaternary structure stability. Cβ-Cβ interactions are observed ∼6 times higher than the known CαHO interaction for helix-helix stabilization. Two additional new interactions of CβO and OO are observed at the AXXXA containing interface regions. The occurrence of the motif gets drastically reduced if any of the terminal Ala residues are replaced by Gly. Our findings show the importance of AXXXA in providing stability to the quaternary structure through specific hydrophobic interactions and the specificity of the Ala residue at motif termini. The knowledge gained can be used for designing synthetic proteins of improved stability and for designing peptide-based therapeutics.

Globular Antifreeze Protein-Inspired Nanoparticle-Based Large-Scale T-Cell Cryoprotection System for Lymphoma Immunotherapy.

Large-scale biosafe T-cell cryopreservation is required to bring T-cell therapies to the market, but it remains challenging due to the cytotoxicity of common cryoprotectants [e.g., dimethyl sulfoxide (DMSO)] and unavoidable ice injuries to cells. Herein, inspired by natural globular antifreeze proteins, we establish a biocompatible zwitterionic magnetic nanoparticle (ZMNP)-based cryoprotection system, achieving large-scale cryopreservation of T cells for lymphoma immunotherapy. ZMNPs could form a globular hydration shell to inhibit water molecule aggregation as well as ice growth, and the surficial hydration strength-antifreeze performance relationship of ZMNPs was investigated. During the thawing process, ZMNPs possessed a magnetic field-mediated nanowarming property that enabled rapid heating and also facilitated easy magnetic separation for cell recovery. These combined effects resulted in a high post-thaw viability (>80%) of large-scale T-cell cryopreservation (20 mL). Notably, post-thaw T cells exhibited similar transcript profiles to fresh cells, while up- or downregulation of 1050 genes was found in the DMSO group. In a mouse E.G7-OVA lymphoma model, ZMNP-system-cryopreserved T cells achieved a tumor suppression rate of 77.5%, twice as high as the DMSO group. This work holds great promise for the application of advanced cryopreservation techniques in the development of therapeutic cellular products.