Babesia bovis is a species of apicomplexan hemoparasitic protozoa that can be transmitted by ticks, causing a global cattle disease. As it depends mainly on the glycolytic pathway for energy production and life cycle maintenance, glycolytic enzymes are possible targets for drug development against Babesia. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) has been one of such targets, against several parasitic organisms. It performs the reversible oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphospho-D-glycerate in the presence of nicotinamide adenine dinucleotide. The protocol for Babesia bovis Glyceraldehyde-3-Phosphate Dehydrogenase (BbGAPDH) expression and purification has been developed to yields of 28 mg of pure protein per liter of culture medium, with a specific activity of 55.5 ± 6.99 U mg-1 after His-tag removal, indicating preservation of enzymatic activity. For crystallization, the His-tag removal proved essential. Crystals diffracted to 3.12 Å resolution in the P3121 space group; NAD+ cofactor molecules are observed in their respective sites. Comparisons to the theoretical model indicate a number of side chain conformation differences. These results provide support for future enzyme inhibition assays, in addition to crystallization assays with potential inhibitors.
While protein A has become a reliable platform for purifying antibodies, an analogous universal purification method for native non-antibody therapeutics has yet to be widely implemented. Downstream processing of recombinant proteins that require a solubility-enhancing domain (SED) for acceptable expression in E. coli are additionally complicated by required proteolytic cleavage for SED removal. This work demonstrates that the Nostoc punctiforme (Npu) DnaE split intein platform can be used for simultaneous purification and SED removal for challenging target proteins. Targets selected for this work include human fibroblast growth factor-2, interleukin-2, and an anti-CTLA-4 nanobody. Each target exhibits improved soluble expression with a solubility-enhancing domain at the N-terminus of the split intein tag, which is fused directly to the N-terminus of the target. Each target was scaled up and purified via FPLC for characterization and activity assays. SUMO-NpuC-FGF-2, MBP-NpuC-IL-2, and SUMO-NpuC-nCTLA4, show at least ≥75% solubility with their respective fusion partners, and could be recovered at up to 98% purity with at least 98% DNA clearance over a single intein column. A single-point mutation in the intein tag was used to further improve the intein cleavage rate for previously difficult target proteins, thereby increasing the appeal of this approach. Activity assays performed on FGF-2 and nCTLA-4 show that this process produces not only highly pure, but also biologically active protein. These results demonstrate a streamlined downstream processing of SED-dependent proteins and also support the potential of intein-mediated purification beyond the discovery phase.
The gap in technical capacity and inadequate infrastructure for biomanufacturing limits progress in vaccine development and biomanufacturing in low- and middle-income countries (LMICs). Peptide subunit antigens offer a promising solution to producing cost-effective, safe and protective vaccines for SARS-CoV-2 and other emerging viral diseases. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which mediates viral entry through interaction with the human angiotensin-converting enzyme 2 (ACE2) receptor, represents a suitable model antigen for building and strengthening local capacity. The aim of this study is to express and validate a recombinant SARS-CoV-2 RBD protein as a vaccine candidate using bacterial expression systems. In this study the RBD sequence is linked to PanDR epitope sequence, an immunostimulant' then codon optimized for bacterial expression. The physicochemical parameter of the protein sequence was assessed using in-silico tools to assess its suitability. The sequence was synthesized chemically, and cloned into a pMAL-c6T expression vector, then transformed into Escherichia coli BL21-DE3 cells. The protein was expressed by IPTG induction for 3 h in Luria-Bertani nutrient media and then purified using affinity chromatography. The recombinant protein was successfully expressed in a bacterial expression system as revealed by a distinct band at the expected molecular weight of approximately 74.5 kDa, on SDS-PAGE corresponding to the MBP-RBD fusion protein. Western blot analysis further confirmed the identity of the protein through specific recognition by anti-MBP antibody. The results show that the modified receptor binding domain protein of SARS-CoV-2 was successfully synthesized, cloned, expressed and validated for further immunological evaluation.
Zika virus (ZIKV) nonstructural protein 1 (NS1) represents a valuable diagnostic target due to its early secretion during infection and reduced cross-reactivity with other flaviviruses. Optimization of NS1 expression conditions is essential for obtaining large-scale protein production. The use of bacterial expression systems as Escherichia coli offers a cost-effective platform. We developed two molecular cloning strategies to express full-length ZIKV NS1 (1053 bp) from Cuban strain 9661/2016 using pQE30/XL1-Blue and pET50b(+)/BL21(DE3) expression systems. The ns1 gene cloned via pGEM-T intermediate vector, and subcloned into expression vectors by using directional restriction-based strategy. The effects of culture medium and IPTG concentration on bacterial growth and protein accumulation was evaluated in order to optimizing the NS1 expression conditions. Both expression systems successfully produced recombinant ZIKV NS1. However, the BL21(DE3)/pET50b(+) system yielded soluble NS1, whereas the XL1-Blue/pQE30 system predominantly produced insoluble protein. Culture medium exerted a greater effect on protein expression than IPTG concentration, with LB medium and 0.1 mM IPTG providing the most favorable conditions for recombinant NS1 production. Notably, XL1-Blue demonstrated competency for protein expression. The pET50b(+)-ns1/BL21(DE3) system offers decisive advantages through soluble expression, eliminating refolding requirements for downstream purification process. This work establishes optimized protocols for ZIKV NS1 expression applicable to obtaining a recombinant protein for developing an ELISA system and broadly transferable to other flavivirus antigens.
Lysozyme can effectively lyse Escherichia coli (E. coli), providing a method for the release of intracellular substances. In this study, an inside-out lysis system based on T4 lysozyme was constructed. Three secretion signal peptides (PelB, NapA, and HybA) were fused to T4 lysozyme to create fusion proteins, among which, NapA-T4 lysozyme (N-T4L) exhibited the highest level of intracellular protein leakage. Microscopic analysis revealed significant cell wall damage in N-T4L expressing cells, confirming the initiation of cell lysis. The influence of chemical factors on cell lysis was also evaluated. Expression of N-T4L reduced cell viability by 2.09 log units following Triton X-100 treatment and pH adjustment. Antibacterial assays demonstrated that N-T4L in the fermentation supernatant maintained substantial inhibitory activity against bacteria. These findings provide a strong foundation for synthetic biology applications involving programmed cell destruction in E. coli.
KIAA1143 is located within the 3p21.3 chromosomal region, which is frequently deleted in human cancers and enriched for tumor suppressor genes, and encodes an evolutionarily conserved protein; however, its biological functions and relevance to tumor biology remain largely uncharacterized. Here, we performed a comprehensive pan-cancer analysis of KIAA1143 through multi-omics data integration, optimized its prokaryotic expression and purification system, constructed its protein interactome by affinity purification coupled with mass spectrometry (AP-MS), and further predicted its three-dimensional structure by using AlphaFold 2. Our results demonstrate that KIAA1143 is widely expressed in multiple human tissues yet exhibits a marked discrepancy between mRNA and protein levels, suggesting stringent post-transcriptional regulation. Pan-cancer analysis further revealed that KIAA1143 is aberrantly expressed across diverse tumors and bears prominent cancer-specific prognostic relevance, exhibiting a bidirectional "prognostic paradox" in different tumor contexts. Interactome profiling identified 73 high-confidence interacting proteins, which were significantly enriched in key cancer-associated pathways, including oxidative phosphorylation, the ubiquitin-mediated proteolysis, mTOR, and MAPK signaling. Structural prediction indicated that KIAA1143 exhibits extensive intrinsic disorder throughout its full-length sequence. This study presents the first systematic multidimensional characterization of KIAA1143 by delineating its pan-cancer expression pattern, prognostic implication, interactome landscape and structural properties, and suggests that KIAA1143 acts as a novel context-dependent regulatory factor in cancer, providing a robust foundation for future mechanistic and functional investigations.
Mature peanut (Arachis hypogaea L.) seeds accumulate abundant superoxide dismutase (SOD), but the high abundance of storage proteins masks trace antioxidant enzymes and prevents accurate profiling of endogenous SOD isoforms by conventional proteomics. Here we report an activity-guided workflow that combines native-PAGE NBT zymography, low-temperature dialysis-bag electroelution and DIA-based nano-LC-MS/MS to enrich native SOD complexes from seed crude extracts. The two SOD-active gel bands, B1 and B2, were excised, embedded in agarose and electroeluted to recover intact proteins before proteomic analysis. Across the crude control (CK) and the two enriched fractions, 1737 non-redundant proteins were annotated, of which 775 (44.6%) were detected only in the SOD-active bands and not in CK, demonstrating efficient enrichment of the hidden proteome. Six SOD-associated proteins were identified: one mitochondrial Mn-SOD, the copper chaperone CCS and four Cu/Zn-SOD isoforms. Quantification showed that the combined abundance of Cu/Zn-SODs exceeded that of Mn-SOD by more than 100-fold, identifying Cu/Zn-SOD as the dominant SOD subtype in mature seeds. Eighteen co-enriched ROS-scavenging enzymes were also detected, and integrative transcriptomics confirmed tissue- and development-specific expression of five of the SOD proteins. This low-cost pipeline with near-quantitative recovery overcomes storage-protein interference and resolves the hidden antioxidant proteome of peanut seeds, providing a general strategy for studying isoenzyme repertoires and redox protein networks in crop seeds.
Engineering mRNA stability is a promising yet underexplored approach for improving recombinant protein production in bacterial systems. In this study, we evaluated the effect of synthetic 3'-UTR hairpin structures on mRNA stability and protein yield in Escherichia coli using two SUMO-fusion expression systems. Hairpin elements with defined structural features were introduced downstream of the coding sequence. In all constructs, 3'-UTR hairpins increased mRNA half-life, with stabilization ranging from approximately 2-fold to 3-fold (n = 3 biological replicates). In the SUMO-SARS-CoV-2-derived peptide system, enhanced transcript stability was accompanied by a marked increase in specific cellular fusion-protein content, reaching up to 6.8-fold relative to the control (n = 3). In the SUMO-liraglutide-derived peptide system, mRNA stabilization was also pronounced, and the increase in specific cellular fusion-protein content reached approximately 3-fold (p < 0.001, n = 6). These findings show that 3'-UTR engineering is an effective strategy for modulating mRNA stability in *E. coli*, but the quantitative relationship between transcript persistence and protein accumulation is context-dependent and likely influenced by additional factors, including translation efficiency. Overall, engineering of 3'-terminal RNA structures provides a practical tool for post-transcriptional tuning of recombinant expression systems.
Age-related macular degeneration (AMD) is a major cause of visual impairment in the elderly, with oxidative stress as its core pathogenesis. Herein, a prokaryotic expression system in Escherichia coli (E. coli) BL21 was developed to yield recombinant human vascular endothelial growth factor B 167(VEGFB167), after systematic optimization of expression and purification parameters, the antioxidant capacity of the target protein was experimentally verified. VEGFB167 was efficiently expressed as inclusion bodies. Following chromatographic purification, the protein purity attained 97% with a concentration of 1.4 mg/mL. Cellular functional assays revealed that VEGFB167 significantly reduced reactive oxygen species, malondialdehyde, and lactate dehydrogenase release, enhanced both superoxide dismutase activity and SOD1/SOD2 gene expression level, inhibited apoptosis, and promoted cell proliferation. These results support further development of VEGFB167 as a biotherapeutic candidate for oxidative injury-triggered illnesses exemplified by AMD.
Nanobodies (Nbs), also known as variable domains of camelid heavy-chain-only antibodies (VHHs), have emerged as promising biomolecules in antiviral diagnostics and therapeutics because of their small molecular size, high stability, strong antigen-binding affinity, and ease of genetic engineering. Compared with conventional monoclonal antibodies (mAbs), Nbs exhibit superior tissue penetration, improved access to cryptic epitopes, cost-effective microbial production, and enhanced physicochemical stability under harsh environmental conditions. These properties make them attractive candidates for applications in viral detection, neutralization, intracellular targeting, imaging, and targeted drug delivery. Recent advances in Nb engineering have enabled the development of multivalent, bispecific, Fc-fused, and aerosolized Nb formats with improved pharmacokinetic and antiviral properties. Nbs have demonstrated promising activity against a broad range of viruses. Furthermore, Nbs have shown considerable utility in biosensors, ELISA systems, live-cell imaging, cryo-electron microscopy, and CRISPR-associated technologies. Despite these advantages, several challenges remain, including short serum half-life, potential immunogenicity, rapid renal clearance, and the need for large-scale clinical validation. This review summarizes the structural and functional characteristics of Nbs, discusses their antiviral mechanisms and engineering strategies, and highlights their expanding applications in viral diagnostics and therapeutics, along with current limitations and future translational perspectives.
The heterologous expression of Bacillus thuringiensis (Bt) Cry toxins in Escherichia coli is frequently hindered by the formation of insoluble inclusion bodies, which severely limits subsequent functional and structural characterizations. In this study, we report an optimized in vitro solubilization and purification protocol utilizing a highly alkaline carbonate buffer (pH 11.3) to efficiently recover Cry toxins. Compared to the conventional method, this optimized strategy significantly enhanced the solubilization efficiency, increasing the yields of Cry1Ab and Cry1Fa by approximately 3.6-fold and 2.75-fold, respectively. Furthermore, the method proved highly effective for the structurally divergent, Coleopteran-specific Cry8Ea1 toxin. Following trypsin activation and size-exclusion chromatography, the purified toxins exhibited high purity and successfully retained their native biological functions. Specifically, purified Cry1Ab and Cry1Fa demonstrated potent receptor-dependent cytotoxicity against Sf9 cells heterologously expressing the BmABCC2 receptor, alongside distinct pore-forming activity in artificial liposomes. Utilizing the high-quality purified Cry8Ea1, we further investigated its oligomerization potential in membrane-mimetic environments, capturing specific early-stage assemblies-including detergent-induced "cloverleaf-shaped" trimers and nanodisc-reconstituted "wedge-shaped" dimers-via negative-stain electron microscopy. Overall, this highly efficient and versatile purification paradigm successfully overcomes the technical bottleneck of inefficient inclusion body solubilization during the in vitro preparation of Cry toxins, thereby laying a reliable material foundation for downstream functional applications and high-resolution structural characterization of their pore-forming mechanisms.
Incomplete removal of the initiator methionine is a frequent bottleneck in Escherichia coli-based production of recombinant proteins, causing heterogeneity and increased immunogenicity of biopharmaceuticals. Methionine aminopeptidase (MAP) is the key enzyme responsible for this post-translational modification, yet its endogenous activity is rapidly saturated under high-level expression conditions. Here, we report a case-study evaluation of a pBR322-derived expression system, previously applied to therapeutic peptides and insulin analogs, in combination with fed-batch cultivation for recombinant production of methionine aminopeptidase in E. coli. The map gene from E. coli BL21(DE3) was cloned into the pF644 vector to generate pF1492. During fed-batch cultivation under the tested conditions, specific productivity reached 127.03 ± 8.66 mg g-1 and volumetric productivity of total cell-associated MAP reached 2.71 ± 0.18 g L-1 by the final hour of induction. MAP accumulated predominantly as insoluble inclusion bodies, which is a common outcome for recombinant protein expression in E. coli at high rates. This study reports upstream production and inclusion body formation only; functional recovery and enzymatic activity were not assessed. Acetate remained moderate (35-60 mM) and biomass was stable, indicating balanced metabolism. The present study evaluates the performance of this system for methionine aminopeptidase as a stress-sensitive model protein under the tested conditions, without a side-by-side comparison with alternative expression systems.
Ewing sarcoma is an aggressive malignancy characterized by the EWS-FLI1 fusion oncoprotein, which remains challenging to target because of its intrinsically disordered nature and lack of intrinsic enzymatic activity. EWSPEP, a peptide derived from the EWS-FLI1 fusion junction region, has previously been investigated in chemically synthesized form; however, its recombinant production has not been reported. In this study, recombinant EWSPEP (rEWSPEP) was expressed in Escherichia coli BL21 Star (DE3) using the pET-45b expression system and purified by Ni2+ affinity chromatography, yielding approximately 25-30 mg/L of bacterial culture with an estimated purity of ∼97% based on SDS-PAGE densitometry. Anti-His Western blotting confirmed the presence of the recombinant product, while MALDI-TOF mass spectrometry showed a predominant ion at m/z 8393.8, corresponding to a molecular mass of approximately 8.4 kDa. Functional evaluation in A673 Ewing sarcoma cells demonstrated a concentration- and time-dependent reduction in cell viability following rEWSPEP treatment, accompanied by pronounced morphological alterations. Conditioned media from treated cells showed increased NPM1 and cleaved PARP immunoreactivity, while Western blot analysis showed decreased NPM1 immunoreactivity in cell lysates following treatment. These findings demonstrate the feasibility of recombinant production of EWSPEP and cytotoxic activity of the purified recombinant preparation in A673 cells under the tested conditions. The established recombinant production platform provides a basis for further structural, mechanistic, and functional characterization of rEWSPEP.
Proteins of similar molecular mass can co-extract, co-elute, and co-migrate during SDS-PAGE, complicating assessment of tissue-derived vimentin preparations. We optimized a self-packed XK16/20 diethylaminoethyl fast-flow (DEAE-FF) workflow for obtaining a porcine brain-derived preparation containing vimentin and added orthogonal identity, composition, and quality-control analyses. Five process variables were evaluated by single-factor experiments and an L16(4^5) design using an equal-weight composite of SDS-PAGE target-band proportion and apparent target-band recovery. Column packing was assessed with an acetone UV254 tracer. The preparation was characterized by bicinchoninic acid assay, SDS-PAGE, anti-vimentin immunoblotting, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and a gel-clot Limulus amebocyte lysate limit test. Trial 9 (pH 7.0, 1:5 solid-to-liquid ratio, 30 min ultrasonication at 430 W, and 1.0 M NaCl in the full-strength elution buffer) produced the highest composite score. The acetone tracer yielded a peak at 12.4 min with an asymmetry factor of 0.944, and the 100% elution step had the highest protein concentration among the evaluated eluates. Three verification batches showed an SDS-PAGE target-band proportion of 89.76 ± 1.82%. LC-MS/MS of the approximately 53-57 kDa gel region identified porcine vimentin (P02543; 33% sequence coverage, 15 peptides, 10 unique peptides, and 18 peptide-spectrum matches) together with multiple co-purifying cytoskeletal protein records. Endotoxin was below assay-derived upper bounds of 0.03 and 0.125 EU/mg. The workflow produced a compositionally heterogeneous, vimentin-containing preparation rather than homogeneous vimentin.
The E3 ubiquitin ligase Nedd4-1 is a structurally complex, multidomain enzyme that plays a crucial role in maintaining proteostasis and regulating the cellular stress response. Nedd4-1's complex regulatory mechanism involves both intermolecular interactions (with upstream E2 conjugating enzymes and substrates) and intramolecular interactions that govern its function. Additionally, Nedd4-1 has received increased attention recently due to a small-molecule N-arylbenzimidazole 2 (NAB2) that prevents proteotoxicity and restores organelle trafficking associated with synucleopathies in a Nedd4-1-dependent manner. To study the enzymology of Nedd4-1, it is essential to employ recombinant Nedd4-1 in its native and untagged form to ensure high-fidelity biophysical characterization. In this study, we develop an efficient and optimized methodology for enhanced expression and purification of untagged active Nedd4-1 from E. coli over three affinity column steps. This strategy employs a nine-residue poly-histidine tag, glutathione S-transferase, and proteolytic cleavage with the TEV fusion protease, His6-MBP-uTEV3, providing 3.9 mg per liter of culture of high-purity (≥95%), active, stable, and storable untagged Nedd4-1. Additionally, the NAB2-Nedd4-1 interaction was re-evaluated using the untagged enzyme via two orthogonal techniques: microscale thermophoresis (MST) and surface plasmon resonance (SPR). Steady-state SPR analysis (χ2 = 0.543) estimated a Kdapp of 169 μM. While binding was constrained by the solubility limits of NAB2, these results suggest that the previously reported nanomolar affinity may be an overestimate resulting from affinity tag interference or immobilization-induced artifacts. This data highlights the potential importance of this method for accessing high-purity, stable untagged Nedd4-1 for biophysical characterization and mechanistic enzymology.
H7N9 subtype avian influenza virus (AIV) is a great threat for poultry industry in China. Serological assays that differentiate infected and vaccinated animals (DIVA) can facilitate H7N9 virus monitoring in vaccinated poultry. A serological method based on a peptide for DIVA diagnosis of H7N9 subtype AIV was previously established. However, complex manufacturing techniques are required for production of chemically-synthesized peptides, casting the affordability and accessibility issues of such assays in poultry farms. Here, to develop an H7N9 DIVA assay suitable for poultry use, a recombinant DIVA peptide fusion protein (DPFP) was expressed in E. coli and its potential as a diagnostic antigen for H7N9 DIVA was assessed. The DPFP composed of the E. coli thioredoxin A, the H7N9 DIVA peptide and His affinity tag was efficiently expressed in a soluble form in E. coli. The DPFP was recognized by H7N9 virus infection serum rather than by H7N9 vaccination serum as determined using immunoblotting. In addition, enzyme-linked immunosorbent assay based on the DPFP was established and optimized, which can well differentiate H7N9 virus infected and vaccinated chickens. OD450 of 0.5 was defined as the cut-off value for H7N9 DIVA diagnosis. Our study indicates that prokaryotic expression system can be used as a promising platform for preparation of diagnostic antigen for discriminating H7N9 virus infected and vaccinated chickens. The method established herein can be employed as an effective and affordable approach for diagnosis and control of H7N9 avian influenza in poultry.
This study aimed to construct a prokaryotic expression vector for the T6SS immunity protein Tldi1 of Salmonella Typhimurium and to prepare its specific polyclonal antibody, thereby facilitating further functional research on Tldi1.The dominant antigenic sequence of Tldi1 was identified through bioinformatics analysis. The target gene was amplified from the genomic DNA of Salmonella Typhimurium SL1344. The recombinant vector pET-32a-tldi1 was then constructed and transformed into Escherichia coli. The recombinant Tldi1 protein was expressed following IPTG induction and purified by nickel affinity chromatography. KM mice were immunized with the purified protein to generate polyclonal antibodies, and the antibody titer and specificity were then determined. The recombinant expression vector was successfully constructed, and the purified Tldi1 protein achieved a purity of over 90%. The prepared polyclonal antibody demonstrated a high titer of 1:256,000and strong specificity. This study successfully obtained high-activity Tldi1 protein and its specific antibody, which provide essential experimental materials for analyzing the T6SS-mediated interbacterial competition mechanism and for establishing a rapid immunological detection method for Salmonella.
Antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics against multidrug-resistant pathogens, but their application is limited by low yield, structural instability, and potential toxicity. In this study, we designed a hybrid peptide, Aur-Defb3, by fusing a bioinformatically optimized rainbow trout beta-defensinderived sequence with a conserved aurein motif. To improve predicted physicochemical properties, we introduced a Leucine-to-Phenylalanine (L-to-F) substitution into the defensin-derived segment, hypothesizing that the aromatic side chain may contribute to hydrophobic-core stability and membrane interaction. Recombinant AurDefb3 was expressed in Komagataella phaffii GS115 and purified by Ni-IDA affinity chromatography. HPLC analysis showed that the major purified peak accounted for 97.08% of the integrated peak area, and the concentration of the purified fraction was estimated by A280 measurement. Antimicrobial assays revealed antibacterial activity against both Gram-positive and Gram-negative bacteria, with MIC values ranging from 31.25 to 250 mu g/mL (average 97.7 mu g/mL). Hemolysis assays showed low hemolytic activity toward rainbow trout erythrocytes under the tested conditions, with approximately 10% hemolysis observed at 256 mu g/mL. Scanning electron microscopy (SEM) showed concentration-dependent morphological damage to bacterial cells, including surface roughening, deformation, and disruption, suggesting that membrane damage may contribute to the antibacterial activity of Aur-Defb3. These results demonstrate the successful expression of Aur-Defb3, supporting its potential for further structural characterization, precise yield quantification, and in vivo mechanistic evaluation.
