Search Results (19)

Komagataella phaffii (formerly Pichia pastoris) is a prominent platform for recombinant protein production, yet secretion efficiency often remains a critical bottleneck. In this study, we validated three candidate genes—WHI3, CLPP, and PMP20—previously identified through genome-wide CRISPR activation screening, for their potential to enhance heterologous protein secretion. Overexpression of these factors under the control of the methanol-inducible AOX1 promoter increased the secretion of human serum albumin (HSA), with WHI3 and CLPP yielding improvements of 18.3% and 17.9%, respectively. Furthermore, applying this strategy to human lactoferrin (hLF) revealed that WHI3 overexpression robustly enhanced hLF secretion by approximately 70%. Comparative analysis of different promoters (AOX1, GAP, and CAT) indicated that the AOX1 promoter remains the most effective driver for these enhancers, suggesting a threshold-dependent regulatory mechanism. These results demonstrate the protein-dependent nature of secretion optimization and identify WHI3, CLPP, and PMP20 as novel, effective co-expression factors for improving recombinant protein yields in K. phaffii. Full article

Background/Objectives: Breast milk lysozyme is crucial for infant intestinal health. The low breastfeeding rate has driven the investigation of alternatives like hen egg white lysozyme (HEWL) for infant formula supplementation. However, HEWL differs significantly from human lysozyme. This study aimed to systematically compare the functional efficacy of recombinant human lysozyme (rhLYZ) and HEWL to assess their suitability as formula supplements. Methods: The physicochemical properties (enzymatic activity, optimal pH, thermal stability) of rhLYZ and HEWL were analyzed. Biological functions were evaluated using HT-29 intestinal cells for proliferation, differentiation, and protection against lipopolysaccharide-induced damage. In vivo effects on growth, intestinal morphology, and gene expression were assessed in a mouse pup model via transcriptomic analysis. Gut microbiota composition was also examined. Results: rhLYZ exhibited twice the enzymatic activity of HEWL, with an optimal pH of 6.0. In cellular models, rhLYZ enhanced intestinal epithelial differentiation at low concentrations. In vivo, rhLYZ supplementation significantly improved pup body weight, intestinal maturity, and villus-to-crypt ratios, outperforming HEWL. Transcriptomics revealed rhLYZ upregulated broad-spectrum antimicrobial peptides (e.g., Defa, lactoferrin) and immune-related genes, whereas HEWL induced a narrower antibacterial response and downregulated key defensins. Furthermore, rhLYZ significantly increased gut microbiota diversity and enriched beneficial butyrate-producing bacteria. Conclusions: rhLYZ more effectively mimics human milk lysozyme by promoting intestinal development, broad-spectrum immunity, and a balanced microbiota. HEWL shows a narrower functional profile. These findings provide a scientific basis for optimizing lysozyme selection in infant formula, highlighting the superior potential of rhLYZ. Full article

Fucoidan is of considerable interest for the development of drug carriers. The inclusion of fucoidan allows calcium carbonate microparticles in the form of vaterite to acquire new properties, enabling their use in the immobilization of protein preparations. In this work, we investigated the properties of hybrid vaterite microparticles with fucoidan from Fucus vesiculosus obtained by co-precipitation and loaded with recombinant human lactoferrin from goats. The hybrid microparticles had a smaller diameter (3–4 µm), larger surface area (35–36 m²g⁻¹), smaller pore size (5–10 nm average), and more negative ζ-potential (−(11–13) mV) than the control vaterite microparticles. The incorporation of lactoferrin into the microparticles by co-precipitation in complex with fucoidan was greater than when the protein was adsorbed onto the hybrid microparticles. Microparticles with fucoidan and lactoferrin were stable in acidic environments, released both components over a prolonged period at pH 7.4, and possessed mucoadhesive properties and anticoagulant activity. The antibacterial properties of hybrid microparticles with fucoidan and lactoferrin against Bacillus subtilis were characterized. Microparticles of vaterite with fucoidan can serve as a platform for the microfabrication of effective means of delivering therapeutic proteins. Full article

The aim of this article was to present the biological activity of milk components, particularly lactoferrin (LF), and techniques for its extraction and purification. Dairy products have long been recognized for their significant contributions to human health and nutrition. Recent studies indicate that dairy consumption offers various health benefits, particularly concerning bone health, metabolic wellness, and cardiovascular health. LF, abundantly present in milk, exhibits a range of health-promoting properties that are increasingly recognized for their significance in nutrition and disease prevention. The production of LF can be approached through two main avenues: extraction from milk and recombinant expression systems. Both methods present unique advantages and challenges that influence the efficiency of LF production on an industrial scale. Moreover, advances in purification and drying techniques are crucial to enhance the overall efficiency of LF production. Recent studies have focused on methods such as monolithic ion-exchange chromatography and membrane technologies to improve yield and reduce costs of LF extraction. These innovations not only facilitate the extraction but also preserve the structural integrity and the functional properties of LF. The article presents the discussion of the applications of the LF in the dairy industry, indicating its growing importance as a functional ingredient in health products. Full article

Lactoferrin (LF) is a major component of human milk. LF supplementation (currently bovine) supports the immune system and helps maintain iron homeostasis in adults. No recombinant human lactoferrin (rhLF) is available for commercial food use. To determine the extent to which rhLF (Effera™) produced by Komagataella phaffii digests similarly to hmLF, a validated in vitro digestion protocol was carried out. Bovine LF (bLF) was used as an additional control, as it is approved for use in various food categories. This study compared the extent of intact protein retention and the profile of peptides released in hmLF, bLF and rhLF (each with low and high iron saturation) across simulated adult gastric and intestinal digestion using gel electrophoresis, ELISA and LC-MS. Intact LF retention across digestion was similar across LF types, but the highest iron-saturated hmLF had greater retention in the simulated gastric fluid than all other sample types. Peptides identified in digested hmLF samples strongly correlated with digested rhLF samples (0.86 < r < 0.92 in the gastric phase and 0.63 < r < 0.70 in the intestinal phase), whereas digested bLF samples were significantly different. These findings support the potential for rhLF as a food ingredient for human consumption. Full article

The effect of lactoferrin on skin was simulated using a recombinant human epidermal model. The anti-inflammatory and soothing effect of lactoferrin was verified using IL-1α and TSLP Elisa assay. The effects of lactoferrin on the expression of related genes and proteins were detected using qPCR and immunofluorescence staining. The results showed that lactoferrin can effectively enhance the Transepidermal Electrical Resistance (TEER) and inhibit the secretion of inflammatory cytokine IL-1α and TSLP. In addition, it was confirmed using qPCR that lactoferrin had high expression levels on AQP3, FLG, IVL, CLDN1 and HAS1 genes. Immunofluorescence staining confirmed that lactoferrin had high fluorescence intensity and expression in AQP3, Filaggrin and Involucrin. The results showed that lactoferrin improved the skin barrier at higher than 1.5 mg/mL. At the same time, it can have anti-inflammatory and moisturizing effects. This study provides a strong basis for the application of lactoferrin in cosmetics and daily chemical products. Full article

DNA polymerases are important enzymes that synthesize DNA molecules and therefore are critical to various scientific fields as essential components of in vitro DNA synthesis reactions, including PCR. Modern diagnostics, molecular biology, and genetic engineering require DNA polymerases with improved performance. This study aimed to obtain and characterize a new CL7-Taq fusion DNA polymerase, in which the DNA coding sequence of Taq DNA polymerase was fused with that of CL7, a variant of CE7 (Colicin E7 DNase) from Escherichia coli. The resulting novel recombinant open reading frame was cloned and expressed in E. coli. The recombinant CL7-Taq protein exhibited excellent thermostability, extension rate, sensitivity, and resistance to PCR inhibitors. Our results showed that the sensitivity of CL7-Taq DNA polymerase was 100-fold higher than that of wild-type Taq, which required a template concentration of at least 1.8 × 10⁵ nM. Moreover, the extension rate of CL7-Taq was 4 kb/min, which remarkably exceeded the rate of Taq DNA polymerase (2 kb/min). Furthermore, the CL7 fusion protein showed increased resistance to inhibitors of DNA amplification, including lactoferrin, heparin, and blood. Single-cope human genomic targets were readily available from whole blood, and pretreatment to purify the template DNA was not required. Thus, this is a novel enzyme that improved the properties of Taq DNA polymerase, and thus may have wide application in molecular biology and diagnostics. Full article

Lactoferrin (LF) stands as one of the extensively investigated iron-binding glycoproteins within milk, exhibiting diverse biological functionalities. The global demand for LF has experienced consistent growth. Biotechnological strategies aimed at enhancing LF productivity through microbial expression systems offer substantial cost-effective advantages and exhibit fewer constraints compared to traditional animal bioreactor technologies. This study devised a novel recombinant plasmid, wherein the AOX1 promoter was replaced with a glucose-inducible G1 promoter (P_G1) to govern the expression of recombinant porcine LF (rpLF) in Pichia pastoris GS115. High-copy-number P_G1-rpLF yeast clones were meticulously selected, and subsequent induction with 0.05 g/L glucose demonstrated robust secretion of rpLF. Scaling up production transpired in a 5 L fermenter, yielding an estimated rpLF productivity of approximately 2.8 g/L by the conclusion of glycerol-fed fermentation. A three-step purification process involving tangential-flow ultrafiltration yielded approximately 6.55 g of rpLF crude (approximately 85% purity). Notably, exceptional purity of rpLF was achieved through sequential heparin and size-exclusion column purification. Comparatively, the present glucose-inducible system outperformed our previous methanol-induced system, which yielded a level of 87 mg/L of extracellular rpLF secretion. Furthermore, yeast-produced rpLF demonstrated affinity for ferric ions (Fe³⁺) and exhibited growth inhibition against various pathogenic microbes (E. coli, S. aureus, and C. albicans) and human cancer cells (A549, MDA-MB-231, and Hep3B), similar to commercial bovine LF (bLF). Intriguingly, the hydrolysate of rpLF (rpLFH) manifested heightened antimicrobial and anticancer effects compared to its intact form. In conclusion, this study presents an efficient glucose-inducible yeast expression system for large-scale production and purification of active rpLF protein with the potential for veterinary or medical applications. Full article

The production of human recombinant proteins to be used for therapeutic or nutritional purposes must focus on obtaining a molecule that is as close as possible to the native human protein. This biotechnological tool has been documented in various studies published in recent decades, with lactoferrin being one of those that has generated the most interest, being a promising option for recombinant technology. However, stability studies including thermodynamic parameters have not been reported for recombinant lactoferrin (Lf). The objective of this work was to obtain the human recombinant protein using the yeast Komagataella phaffii to study structural changes modifying pH and temperature using circular dichroism spectroscopy (CD). Thermodynamic parameters such as ΔH, ΔS and Tm were calculated and compared with commercial human lactoferrin. We propose the potential use of CD and thermodynamic parameters as a criterion in the production of recombinant proteins to be used in the production of specialized recombinant proteins. Full article

Laboratory production of recombinant mammalian proteins, particularly antibodies, requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications. Transient gene expression (TGE) in the suspension-adapted Chinese Hamster Ovary (CHO) cell lines has become the method of choice for this task. The antibodies can be secreted into the media, which facilitates subsequent purification, and can be glycosylated. However, in general, protein production in CHO cells is expensive and may provide variable outcomes, namely in laboratories without previous experience. While achievable yields may be influenced by the nucleotide sequence, there are other aspects of the process which offer space for optimization, like gene delivery method, cultivation process or expression plasmid design. Polyethylenimine (PEI)-mediated gene delivery is frequently employed as a low-cost alternative to liposome-based methods. In this work, we are proposing a TGE platform for universal medium-scale production of antibodies and other proteins in CHO cells, with a novel expression vector allowing fast and flexible cloning of new genes and secretion of translated proteins. The production cost has been further reduced using recyclable labware. Nine days after transfection, we routinely obtain milligrams of antibody Fabs or human lactoferrin in a 25 mL culture volume. Potential of the platform is established based on the production and crystallization of antibody Fabs and their complexes. Full article

In this article, we review the benefits of applying bovine colostrum (BC) and lactoferrin (LF) in animal models and clinical trials that include corticosteroid application and psychic stress, treatment with non-steroid anti-inflammatory drugs (NSAIDs) and antibiotics. A majority of the reported investigations were performed with native bovine or recombinant human LF, applied alone or in combination with probiotics, as nutraceutics and diet supplements. Apart from reducing adverse side effects of the applied therapeutics, BC and LF augmented their efficacy and improved the wellness of patients. In conclusion, LF and complete native colostrum, preferably administered with probiotic bacteria, are highly recommended for inclusion in therapeutic protocols in NSAIDs and corticosteroid anti-inflammatory, as well as antibiotic, therapies. These colostrum-based products can also be of value for individuals subjected to prolonged psychophysical stress (mediated by endogenous corticosteroids), especially at high ambient temperatures (soldiers and emergency services), as well as physically active people and training athletes. They are also recommended for patients during recovery from trauma and surgery, which are always associated with severe psychophysical stress. Full article

In this article, we review the benefits of application of colostrum and colostrum-derived proteins in animal models and clinical trials that include chemotherapy with antimetabolic drugs, radiotherapy and surgical interventions. A majority of the reported investigations was performed with bovine colostrum (BC) and native bovine or recombinant human lactoferrin (LF), applied alone, in nutraceutics or in combination with probiotics. Apart from reducing side effects of the applied therapeutics, radiation and surgical procedures, BC and LF augmented their efficacy and improved the wellness of patients. In conclusion, colostrum and colostrum proteins, preferably administered with probiotic bacteria, are highly recommended for inclusion to therapeutic protocols in cancer chemo- and radiotherapy as well as during the surgical treatment of cancer patients. Full article

Lactoferrin (LF), known to be present in mammalian milk, has been reported to promote the proliferation of osteoblasts and suppress bone resorption by affecting osteoclasts. However, the mechanisms underlying the effects of human sources LF on osteoblast differentiation have not yet been elucidated, and almost studies have used LF from bovine sources. The presented study aimed to characterize the molecular mechanisms of bovine lactoferrin (IF-I) and human recombinant lactoferrin (LF-II) on MC3T3-E1 pre-osteoblast cells. MC3T3-E1 cells were treated with LF, ascorbic acid, and β-glycerophosphate (β-GP). Cell proliferation was analyzed using the MTT assay. Alkaline phosphatase activation and osteopontin expression levels were evaluated via cell staining and immunocytochemistry. The differentiation markers were examined using quantitative real-time PCR. The cell viability assay showed the treatment of 100 μg/mL LF significantly increased; however, it was suppressed by the simultaneous treatment of ascorbic acid and β-GP. Alizarin red staining showed that the 100 μg/mL treatment of LF enhanced calcification. Quantitative real-time PCR showed a significant increase in osterix expression. The results suggest that treatment with both LFs enhanced MC3T3-E1 cell differentiation and promoted calcification. The mechanisms of calcification suggest that LFs are affected by an increase in osterix and osteocalcin mRNA levels. Full article

We have recently developed probiotics that can express bovine, human, or porcine lactoferrin (LF), and the present study evaluated the effect of these probiotics in improving non-alcoholic fatty liver disease (NAFLD). Three kinds of probiotic supplements, including lactic acid bacteria (LAB), LAB/LF, and inactivated LAB/LF, were prepared. The LAB supplement was prepared from 10 viable LAB without recombinant LF-expression, the LAB/LF supplement was prepared from 10 viable probiotics expressing LF, and the inactivated LAB/LF supplement was prepared from 10 inactivated probiotics expressing LF. A model of obese/NAFLD mice induced by a high-fat diet was established, and the mice were randomly divided into four groups and fed with a placebo, LAB, LAB/LF, or inactivated LAB daily for four weeks via oral gavage. The body weight, food intake, organ weight, biochemistry, and hepatic histopathological alterations and severity scoring were measured. The results revealed that the obese mice fed with any one of the three probiotic mixtures prepared from recombinant probiotics for four weeks exhibited considerably improved hepatic steatosis. These findings confirmed the assumption that specific probiotic strains or LF supplements could help to control NAFLD, as suggested in previous reports. Our data also suggest that the probiotics and LFs in probiotic mixtures contribute differently to improving the efficacy against NAFLD, and the expressed LF content in probiotics may help to boost their efficacy in comparison with the original probiotic mixtures. Moreover, when these LF-expressing probiotics were further inactivated by sonication, they displayed better efficacies than the viable probiotics against NAFLD. This study has provided intriguing data supporting the potential of recombinant probiotics in improving hepatic steatosis. Full article

Lung cancer continues to be the deadliest cancer worldwide. A new strategy of combining chemotherapeutics with naturally occurring anticancer compounds, such as lactoferrin, might improve the efficacy and toxicity of current chemotherapy. The aim of this study was to evaluate the effect of recombinant human lactoferrin (rhLf) in combination with etoposide on anticancer activity in human lung adenocarcinoma cells. In addition, we examined the impact of rhLf on etoposide-induced cytotoxicity of human endothelial cells. We found that treatment of A549 cells with a combination of etoposide and rhLf resulted in significantly greater inhibition of cancer cell growth as compared to etoposide alone. The combination repressed cancer cell growth by cell cycle arrest in the G2/M phase and induction of apoptosis. In contrast to cancer cells, rhLf did not affect endothelial cell viability. Importantly, rhLf significantly diminished the etoposide-induced cytotoxicity of endothelial cells. Analysis of the type of drug interaction based on combination index value showed that rhLf synergized with etoposide to induce anticancer activity. The calculated dose reduction index indicated that the combination treatment reduced a 10-fold of etoposide dose to achieve the same anticancer effect. Our data demonstrate that rhLf enhanced the anticancer activity of etoposide and diminished etoposide-induced cytotoxic effect in endothelial cells. Full article