The rapid emergence of antimicrobial resistance has intensified the demand for novel anti-infective agents, positioning antimicrobial peptides (AMPs) as promising therapeutic alternatives. However, large scale production of AMPs remains challenging due to their small size, cationic nature, host toxicity, and high cost of chemical synthesis. In this study, we report an efficient and economically viable intein-mediated recombinant bioprocess for the production of the short antimicrobial peptide Lasioglossin-III (LL-III) in its C-terminally amidated form using Escherichia coli-based expression platform. A codon-optimized LL-III gene was fused to the Mxe GyrA intein along with a chitin binding domain (CBD) using the pTWIN1 expression system, enabling stable expression, affinity purification, and controlled auto-cleavage. The fusion protein was expressed in soluble form at low temperature and purified using chitin resin under optimized binding conditions. Notably, the chitin resin retained its binding capacity across multiple reuse cycles, improving the overall process-economics; auto-cleavage under mild reducing conditions released the mature peptide, which was confirmed by RP-HPLC and LC/MS to be identical to chemically synthesized standard peptide. The optimized process yielded ∼4.4 mg L-1 of recombinant peptide leading to significant reduction in production costs at the current cleavage efficiency. Overall, this study establishes a scalable, environmentally sustainable, and cost-effective bioprocessing platform not only for short AMPs, but also for other peptide-based therapeutics as well.

Circulating tumor cells (CTCs) are important biomarkers of cancer metastasis; however, their extremely low abundance in the bloodstream poses significant challenges for sensitive and specific detection. Bifunctional fusion proteins that combine an IgG-binding domain with a signal-amplifying module offer a promising strategy to address this challenge. In this study, we constructed a recombinant bifunctional fusion protein, designated r-Protein G-SA, by fusing four tandem repeats of the high-affinity C3 domain of streptococcal Protein G to core streptavidin via a flexible (GGGGS)3 linker. The purified protein was obtained in high purity as assessed by SDS-PAGE and adopted a well-defined secondary structure as determined by circular dichroism (CD) spectroscopy. Surface plasmon resonance (SPR) analysis revealed a high apparent binding affinity for human IgG, with an apparent Kd of 13.98 nM. In immunofluorescence assays, the fusion protein enabled clear discrimination among SKBR3 (HER2 3+), ZR-75-1 (HER2 2+), and MCF-7 (HER2 1+) cells, whereas no detectable signal was observed in HER2-negative MDA-MB-231 and white blood cells (WBCs). In spiked rare-cell capture experiments (5, 50, 500, 1,000, or 2000 SKBR3 cells spiked into 10 mL blood), recovery rates exceeded 80% with good linearity (R[2] = 0.9994). Collectively, the r-Protein G-SA fusion protein retains both IgG Fc-binding and biotin-binding activities, enabling sensitive and specific labeling of target cells. These results demonstrate its potential as a versatile tool for antibody-based detection and rare-cell enrichment. Nevertheless, validation in clinical patient samples is required prior to any clinical application.

Tuberculosis remains among the top ten deadliest infectious diseases worldwide, primarily due to delayed diagnosis, co-infection with HIV, and multidrug resistance. Targeting early secreted mycobacterial antigens, including MPT64, ESAT6, and CFP10, may be a viable strategy for detecting the onset of tuberculosis. Although recombinant MPT64 has been produced previously, limited information is available regarding its structural integrity and thermodynamic unfolding behaviour following recovery from inclusion bodies. In this study, MPT64 protein was overexpressed, affinity-purified, and characterised using biophysical and biochemical methods. The purified recombinant MPT64 had a purity greater than 95%, yielding 2 mg from 1 L of bacterial culture. Western blot analysis confirmed MPT64 identity, while ELISA demonstrated antibody recognition of refolded MPT64, with markedly reduced binding following heat denaturation, indicating that refolding preserved conformational epitopes. Far-UV circular dichroism (CD) and intrinsic fluorescence spectroscopy spectral profiles confirmed that the purified MPT64 protein retained its folded secondary and tertiary structural integrity. Thermodynamic analysis by fluorescence and Far-UV CD yielded stability values (ΔG°) of 19.9 and 26.2 kJ mol[-1], with unfolding midpoints of 5.3 and 5.5 M urea, respectively. Differential scanning calorimetry and thermal circular dichroism indicated melting transitions between 62.5 and 58 °C, supporting moderate thermal stability. Collectively, these complementary measurements provide a detailed biophysical assessment of the conformational and thermal stability of recombinant MPT64 following inclusion-body recovery and refolding. The findings provide a biophysical basis for further investigation of MPT64 as a recombinant antigen for tuberculosis diagnostics.

Omicron variants of the SARS-CoV-2 Spike protein have been challenging to produce recombinantly at high yields compared to their wild type counterparts. Using a variety of mutants within the receptor-binding domain of Spike, we have been able to identify a specific mutation, S375F, in Omicron variants which leads to the reduced protein production yields, likely due to decreased expression and reduced protein stability. In addition to revertant mutations which can improve the yield of Omicron proteins, we also demonstrate that a new HEK293 expression system, Gibco™ Expi293™ PRO, can restore higher yield for these challenging variants, eliminating the need for additional mutations.

Interleukin-40 (IL-40) is a novel cytokine involved in IgA production and B-cell development. Currently, the investigation of recombinant human IL-40 (rhIL-40) is limited to functional studies. In this study, we expressed rhIL-40 and optimized conditions in the methylotrophic yeast, Komagataella phaffii. We introduced the pPICZαA plasmid containing a mature human IL-40 into K. phaffii KM71H by electroporation, screened the transformants on Zeocin™ gradients, and confirmed gene integration by PCR. The secretory expression level of rhIL-40 was approximately 187.30 mg/L after 48 h of induction by 1% (v/v) methanol and addition of phenylmethylsulfonyl fluoride and pepstatin A to the culture medium at 28 °C. Interestingly, rhIL-40 was high molecular weight with N-linked glycans, indicating reduction of the molecular weight to the expected 28 kDa after PNGase F digestion. We also investigated the effect of N-linked glycosylation on rhIL-40 expression and secretion. A single asparagine mutation (N86A or N132A) negatively affected rhIL-40 expression and secretion levels. Moreover, the double mutation revealed a dramatic reduction in rhIL-40 expression and secretion, with little accumulation of the expected intracellular non-glycosylated rhIL-40, suggesting that N-glycosylation mainly influenced the stability of rhIL-40, leading to a lower level of secretory rhIL-40. Our results indicate the feasibility of using K. phaffii KM71H for rhIL-40 expression and the importance of N-linked glycosylation for rhIL-40 stability in the yeast expression system.

The phytopathogenic fungus Chrysoporthe cubensis was cultivated on agro-industrial residues to evaluate laccase production, with coffee husks inducing the highest activity. The enzyme, identified as laccase MCO12, was purified and biochemically characterized, showing acidic activity (pH 2.2-5.0), optimum at 55 °C, and stability between 45 and 60 °C, with half-lives of 290, 22, and 3 min at 45, 55, and 65 °C, respectively. To improve yield and stability, the MCO12 gene was codon-optimized and overexpressed in Komagataella phaffii, resulting in 246 U/L of recombinant enzyme. The recombinant laccase displayed superior thermal performance, retaining >80% activity from 55 to 70 °C, with an optimum at 60 °C, and extended half-lives of 142, 45, and 11 h at 50, 60, and 70 °C, respectively. Molecular docking with ABTS revealed stronger binding affinity and stabilization for the recombinant form, supporting enhanced substrate specificity. Functionally, recombinant MCO12 achieved high dye decolorization efficiency, removing 99% of Malachite Green and 71% of Congo Red at 50 °C after 72 h, without chemical mediators. Overall, these findings demonstrate that recombinant MCO12 is a robust and thermostable laccase, with promising potential for industrial and environmental applications, particularly in the bioremediation of dye-contaminated effluents from the textile industry.

The end-to-end characterization of a fed-batch bioprocess for expressing a non-originator version of the NISTmAb IgG1 (cNISTmAb) using NISTCHO, a high-performance CHO production cell line available as a NIST reference material (RM 8675), is described. NISTCHO was developed to support bioprocess reproducibility, cross-laboratory standardization, technology benchmarking, and collaborative innovation in biomanufacturing process research. Propagation, scale-up, and cultivation of NISTCHO in fed-batch mode using 3 L single-use bioreactors is shown to yield reproducible titers exceeding 2 g/L. Upstream cell culture performance demonstrated predictable growth kinetics, sustained viability, and stable cell morphology across independent bioreactor runs. Metabolite analysis revealed characteristic CHO behavior, including lactate accumulation followed by consumption during later culture stages. Downstream purification consisting of Protein A (proA) capture followed by sequential anion exchange and cation exchange chromatography enabled efficient removal of host cell proteins and nucleic acids, producing high-purity antibody with overall recoveries of (62 to 70) %. Fc glycosylation analysis demonstrated a profile dominated by core fucosylated and galactosylated species, with low levels of high-mannose and α2,6-sialylated glycans, consistent with a typical CHO-derived IgG1 product. Together, these results establish a reproducible upstream and downstream bioprocess for high-yield, high-purity cNISTmAb using NISTCHO.

Sugarcane smut, caused by the biotrophic fungus Sporisorium scitamineum, represents a devastating global threat to sugarcane production. During fungal infection, secreted chitinases are involved not only in cell wall remodeling and nutrient acquisition, but also in the suppression of host immune recognition, making chitinases attractive targets for antifungal drug development. Despite their importance, the catalytic and inhibitory mechanisms underlying chitinases from S. scitamineum remain poorly understood. Here, we report the crystal structure of the S. scitamineum chitinase SsChiA at a resolution of 1.84 Å. SsChiA adopts a canonical (β/α)8 TIM-barrel catalytic domain coupled to a chitinase insertion domain (CID) characteristic of CID-containing GH18 chitinases, together forming a pronounced substrate-binding groove. Structural analysis revealed that the N-terminal His-tag peptide of a symmetry-related molecule specifically bound within this groove, which might suggest a competitive inhibitory mechanism mediated by occupancy of the catalytic site. Site-directed mutagenesis confirmed that Asp286 and Glu288 within the conserved DxDxE motif are important for enzymatic activity. Combining structural analysis with molecular docking of an SsChiA-chitinopentaose complex model, we propose that SsChiA employs the conserved substrate-assisted retention mechanism characteristic of the GH18 family. Furthermore, on the basis of the asymmetric distribution of aromatic residues in the active center and structural comparisons with known exochitinases, we hypothesize that SsChiA functions as a nonreducing-end exochitinase. This study provides the first mechanistic characterization of the S. scitamineum chitinase SsChiA, establishing a structural framework for the rational design of environmentally friendly fungicides targeting this enzyme.

Bifidobacterium species preferentially accumulate in solid tumors and have emerged as a promising platform for localized production of therapeutic molecules within the tumor microenvironment. In this study, we constructed a recombinant B. longum strain capable of secreting an anti-mouse programmed cell death-1 (PD-1) single-chain variable fragment (scFv) and evaluated its expression, antigen-binding activity, blocking activity, and binding kinetics. Purified anti-mouse PD-1 scFv was detected as a ∼27 kDa protein by SDS-PAGE and confirmed by Western blot analysis. The antigen-binding activity of the purified scFv was evaluated by ELISA using recombinant mouse PD-1, yielding an apparent dissociation constant (Kd) of 0.053 μg/mL (1.9 nM). Furthermore, an in vitro PD-1/PD-L1 blocking assay demonstrated that preincubation of immobilized PD-1 with the scFv resulted in a concentration-dependent reduction in PD-L1 binding at PD-L1 concentrations of 0.03, 0.1, and 0.3 μg/mL, with relative IC50 values of 0.58, 0.96, and 1.59 μg/mL, respectively. Although PD-L1 binding was inhibited in a concentration-dependent manner, residual binding remained even at the highest scFv concentration tested. Biolayer interferometry further showed concentration-dependent binding of the purified scFv to mouse PD-1. Collectively, these findings demonstrate that B. longum can produce an anti-mouse PD-1 scFv with in vitro binding and blocking activities and provide a foundation for the future development of bacteria-mediated localized immune checkpoint blockade for solid tumors.

Efficient purification of rat-derived antibodies is an ongoing challenge in biologics development, particularly for isotypes that are difficult to capture using conventional affinity methods. Traditional Protein A/G affinity resins often exhibit limited efficiency and scalability for rat antibodies, particularly rat IgG1, which binds poorly to these ligands. Aside from efficiency and scalability concerns, broad application of affinity resins can be limited by sodium hydroxide ligand incompatibility. To address these limitations, we developed two alternative purification processes tailored to different production scales. For small-scale discovery screening, CaptureSelect IgG-Fc significantly outperformed Protein G across three rat IgG1 monoclonal antibodies, achieving monomer yields of 62.7-89.9% (compared to 32.1-43.1% for Protein G) and purities exceeding 90% in a straightforward bind-and-elute operation. For gram-scale production, we established a sanitizable multi-step process utilizing POROS 50 HS cation-exchange capture followed by Capto Q flow-through anion-exchange polishing. Design-of-experiments optimization of the Capto Q step (pH 7.4-7.6, 50-60 mM Tris-HCl) delivered over 90% monomer purity and overall process recoveries of 59.9-76.6%. ELISA assays verified antibody integrity and binding activity for both methods. Together, these results provide a practical process selection framework that expands production options for hybridoma-derived antibodies.

Protein L is a κ light-chain-binding protein, which is widely used as an affinity ligand for antibody purification and characterization. However, manufacturable recombinant Protein L ligands remain insufficiently studied, particularly regarding how domain architecture affects production, processability, and product quality. In the current study, we designed, expressed and purified three recombinant Protein L variants with distinct domain architectures. This allowed the influence of domain architecture on manufacturability, stability, and downstream functionality to be explored. While differences in intermediate purity and recovery were observed among the three different variants, the optimized process allowed highly purified recombinant Protein L to be generated. As a functional validation, one of the purified variants (i.e., rPL02) was immobilized onto a membrane support, which was used to purify a κ light chain-containing single-chain variable fragment (scFv). The Protein L membrane device not only captured the target scFv but also removed the product-related aggregates under the tested conditions. In conclusion, the current work established a practical platform for producing functional recombinant Protein L. Meanwhile, it provided valuable information for future development of manufacturable recombinant Protein L for affinity media.

Matrix metalloproteinase-13 (MMP-13), a key regulator of extracellular matrix remodeling, has been implicated in tumor invasion and metastasis. This study investigated MMP-13 expression in canine mammary carcinomas and evaluated its diagnostic potential. MMP-13 mRNA and protein expression were assessed by quantitative real-time PCR and immunohistochemistry, respectively. Compared with healthy mammary tissues (n = 10), CMT tissues (n = 27) exhibited a significant 6.63 ± 0.70-fold upregulation of MMP-13 transcripts (p ≤ 0.05). A recombinant canine MMP-13 was produced (55 kDa approx.), functionally validated by zymography and cell invasion assay, and used to develop a sandwich ELISA. The assay demonstrated excellent diagnostic performance, distinguishing tumor-bearing dogs from healthy controls with an area under the receiver operating characteristic curve of 0.968, 96.30% sensitivity, and 88.60% specificity. Immunohistochemistry showed strong MMP-13 expression in 70.37% of canine mammary carcinomas. These findings demonstrate that MMP-13 is overexpressed in canine mammary carcinomas and suggest that MMP-13 protein has the potential to serve as an additional candidate biomarker for the detection of canine mammary carcinomas. However, further validation in a larger set of samples is warranted.

Astaxanthin biosynthesis in engineered bacteria commonly relies on CrtW/CrtZ enzymes, whereas functional reconstruction of the yeast P450-type CrtS/CrtR module in Escherichia coli remains less established. Here, crtS and crtR from Xanthophyllomyces dendrorhous were introduced into pBR322-derived vectors and co-expressed with pAC-BETAipi in E. coli DH5α. The parental pAC-BETAipi strain and the CrtS/CrtR co-expression strain showed comparable growth profiles. HPLC analysis of the co-expression strain revealed an astaxanthin-associated target peak at 2.734 min. After addition of the astaxanthin standard to the same extract, the target-peak area increased from 448,930 to 812,037 while the retention time remained essentially unchanged (2.734 versus 2.732 min). Among five carbon sources, maltose gave the highest wet-biomass-normalized target-peak yield (16.28 ± 0.29 μg/g; P < 0.05). Under a nominal C/N ratio of 6:1, product selectivity reached 73.8%. These results support functional compatibility of the yeast CrtS/CrtR module with a bacterial β-carotene background and identify culture conditions favoring the astaxanthin-associated target peak.

TOYOPEARL NH2-750F is a salt tolerant anion exchange (AEX) resin from Tosoh, which is designed for intermediate purification of mAbs and other types of biomolecules. It is composed of polymethacrylate beads that have been functionalized with primary amine (NH2) AEX groups. Unlike conventional AEX resins, NH2-750F resin can maintain its capacity at conductivities up to 15 mS/cm when used in bind-elute mode. In a sense, NH2-750F is similar to mixed-mode media as both electrostatic and hydrophobic interactions contribute to protein retention. A remarkable difference between NH2-750F and conventional AEX also lies in the fact that certain proteins can bind to the former but not the latter at pHs below their isoelectric points (pIs). According to its manufacturer, NH2-750F shows improved selectivity for protein monomer. In the current work, with two case studies, we demonstrate that NH2-750F possesses unique protein-binding property and good byproduct separation potential, which distinguishes itself from conventional AEX and AEX-dominated mixed-mode resins. This resin's strong byproduct separation capability and moderate price make it an attractive tool for intermediate and polishing purification.

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.