• Food Science of Animal Resources
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• v.21 no.4
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• pp.374-382
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• 2001

Lactoferrin(Lf) has the function of modulation in the host defense systems, including cytokine production and immune responses. We have tested the effect of Lf and Lf ydrolysates(Lf-H) on the productions of tumor necrosis factor-${\alpha}$(TNF-${\alpha}$) and nitric oxide(NO) in macrophage cells. Lf from Korean native cattle(K-Lf) and hydrolyzed K-Lf(K-Lf-H) increased the production of TNF-${\alpha}$ in RAW264.7 cells with dose-dependency. Bovine Lf(B-Lf), human Lf(H-Lf), and its hydrolysates did not induce either TNF-${\alpha}$ production or NO production. On the other hand those didn\`t affect on the production of TNF-${\alpha}$ in lipopolysaccharide(LPS)-stimulated RAW264.7 cells. K-Lf induced the production of NO similar to its role on the TNF-${\alpha}$ production.

• Korean Journal of Agricultural Science
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• v.23 no.1
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• pp.80-89
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• 1996

The expression and secretion of human lactoferrin (hLf) in Sacclnromyces diastaticus were performed. 1. For the secretion of hLf in yeast, recombinant plasmid pYEGLf was constructed using promoter, secretion signal sequence of glucoamylase I gene (STA1) and transcriptional terminator of GAL7 gene. 2. Each correct recombinant plasmid was selected by mini-preparation of plasmid DNA from E coli transformant and restriction enzyme digestion analysis. The selected plasmids, pYEGLf, were transformed into S. diastaticus YIY345 as a expression host, respectively. 3. Western blot analysis using rabbit anti-hLf was carried out to identify expressed hLf. Positive signals were shown in culture supernatant of pYEGLf transformant. 4. About $100{\mu}g-1mg$ of concentrated culture supernatant of positive clone were loaded on paper disc and tested for the antimicrobial activity against E coli. However, no activity was observed. We concluded that this fact results from low concentration of hLf secreted from yeast, compared with the fact that MIC of hLf is as high as $3mg/m{\ell}$. Therefore, the purification of secreted hLf may be require to investigate the antimicrobial activity. From this study, the feasibility of low-cost production of sufficient quantities of human lactofferin for nutritional and therapeutical applications were suggested.

• Korean Journal of Microbiology
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• v.51 no.3
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• pp.271-279
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• 2015

Lactoferrin (LF) is a multifunctional, iron-binding glycoprotein found in physiological secretions of mammals. LF shows antibacterial, antiviral and antifungal activities. In the present study, a gene encoding the N-terminal lobe of human lactoferrin (hLF) was isolated, cloned and expressed in methylotrophic yeast, Pichia pastoris. The recombinant hLF-N (rhLF-N) protein was secreted into the culture medium at the level of $458{\mu}g/ml$ in 3 L fermentor. The size of purified hLF-N was estimated as 35 kDa when analyzed by SDS-PAGE and western blotting. The rhLF-N was further confirmed by immunodiffusion using the anti-hLF polyclonal antibody. The expression profile analysis by qRT-PCR showed that the relative mRNA expression of rhLF-N was maximal after 2-3 days of methanol induction and reduced gradually at 4 days. The purified rhLF-N showed broad antibacterial activities against the pathogens such as Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, Burkholderia cepacia, and Salmonella typhimurium. However, rhLF-N showed relatively lower activity when compared to peptides derived from LF. In spite of this weak activity, the rhLF-N expressed in P. pastoris might be more advantageous for the industrial application, because rhLF-N is secreted into the culture medium and the production can also be increased by optimization of culture conditions.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.5
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• pp.442-448
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• 2006

Human lactoferrin (hLf) is an iron-binding glycoprotein that has been considered to play many biological roles in the human, including the stimulation of the immune system, antimicrobial and anti-inflammatory effects, and regulation of iron absorption. We generated transgenic Siberian ginseng (Acanthopanax senticosus) cell cultures producing a functional hLf protein using the signal peptide sequence from the endoplasmic reticulum and driven by an oxidative stress-inducible SWPA2 promoter which is highly expressed in plant cell cultures. The production of hLf increased proportionally to cell growth and showed a maximal level (up to 3.6% of total soluble protein) at the stationary phase in suspension cultures. Full-length hLf protein was identified by immunoblot analysis in transgenic cell cultures of Siberian ginseng. Recombinant hLf (rhLf) was purified from suspension cells of Siberian ginseng by ammonium sulfate precipitation, cation-exchange and gel filtration chromatography. N-terminal sequences of rhLf were identical to native hLf (nhLf). The overall monosaccharide composition of rhLf showed the presence of plant specific xylose while sialic acid is absent. Antibacterial activity of purified rhLf was higher than that of nhLf. Taken together, we anticipate that medicinal Siberian ginseng cultured cells, as demonstrated by this study, will be a biotechnologically useful source for commercial production of functional hLf not requiring further purification.

• Reproductive and Developmental Biology
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• v.32 no.1
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• pp.45-49
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• 2008

We have previously produced transgenic (TG) mice expressing the human lactoferrin (hLF), interleukin-10 (hIL-10), and thrombopoietin (hTPO) proteins in the milk. In this study, we examined whether simple crossbreeding between two kids of a single transgenic mouse can produce double transgenics co-expressing two human proteins.. The hLF male, and the hIL-10 male were crossbred with the hIL-10 and hTPO females, and the hTPO female, respectively. PCR analysis for genotyping showed 32%, 23% and 24% double transgenic rates for hLF/hIL-10, hLF/hTPO, and hIL-10/hTPO transgenes, respectively. We analyzed the expression levels of the human proteins from double transgenic mice and compared those with their single transgenic siblings. All double transgenic co-expressed two human proteins at comparable levels to singles', unless hTPO was not co-expressed: for hLF, 1.1 mg/ml in hLF/hIL-10, whereas 0.5 mg/ml in hLF/hTPO; for hIL-10, 4.1 mg/ml in hIL-10/hLF, whereas 1.4 mg/ml in hIL-10/hTPO. Ihe downregulation of hTPO to half level of singles' was observed in double transgenic mice. The possible reason why hTPO co-expressed might lead to down-regulation of another human protein was discussed. These results suggested that double transgenic generated by crossbreeding between two singles' could be useful system for bioreactor.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.85-85
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• 2003

Human lactoferrin (hLF) is an 80 kDa iron-binding glycoprotein that is expressed in high concentration in milk and in lesser amount in the secondary or specific granules of neutrophils and in plasma, LF is classically considered to be related to the binding, transport, and storage of iron. The transgenic mice carrying the human hLF gene in conjunction with the bovine $\beta$-casein promoter produced the human hLF in their milk during lactation. To screen transgenic mice, PCR was carried out using chromosomal DNA extracted from tail or toe tissues. In this study, stability of germ line transmission and expression of hLF were monitored up to generation Fl7 of a transgenic line. When female mouse of generation F9 was crossbred with normal male, generation F9 to Fl7 mice showed similar transmission rates ($66.0 \pm 12.57%, 42.0 \pm 14.98%, 72.2 \pm 25.45%, 50.0 \pm 16.70%, 65.7 \pm 6.45%, 48.6 \pm 14.65%, 54 1 \pm 18 11%, 57.8 \pm 16.16% and 48.6 \pm 20.66$, respectively), implying that the hLF gene can be transmitted stably up to long term generation in the transgenic mice For ELISA analysis, hLF expression levels were determined with an hLF ELISA kit in accordance with the supplier's protocol. Expression levels of human hLF from milk of generation F9 to Fl3 mice were $ 3.2 \pm 0.69 mg/ml, 3.1 \pm 0.81 mg/ml, 4.6 \pm 1.38 mg/ml, 3.1 \pm 0.42 mg/ml, and 4.5 \pm 1,48 mg/ml$, respectively. These expression levels were lower than that of founder (6.6 mg/$m\ell$) mouse. We concluded that transgenic mice faithfully passed the transgene on their progeny and successively secreted target proteins into their milk through several generations.

• Korean Journal of Microbiology
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• v.40 no.4
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• pp.348-354
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• 2004

The expression and antibacterial. activity of recombinant human lactoferrin (hLf) was studied from meth­ylotrophic yeast Pichia pastoris. The gene encoding hLf, isolated from human breast cDNA library, was subcloned into the expression vector, pPIC3.5K under the control of AOX1 promoter. The gene was integrated into the host chromosome and was identified by Southern blotting. The expression of the integrated gene was investigated by RT-PCR, Northern blotting, SDS-PAGE and Western blotting. Discrete band corresponding to hLf was detected from the SDS-PAGE, which was confirmed by Western blotting. The expression was also confirmed by RT-PCR and Northern blotting. The antibacterial activity of the recombinant hLf (rhLf) was investigated using Staphy­lococcus aureus ATCC 6538P and Micrococcus flavus ATCC 10240 as test organisms. The rhLf showed strong antibacterial activities against the bacteria. Furthermore, many Gram-negative animal pathogens such as E.coli ATCC8739, 25922, and Salmonella typhimurium 114 and 115, Pseudomonas fluorescens ID 963 I, P. aeruginosa KCCM 11802, and Gram-positive bacteria Bacillus mesentericus were also inhibited in their growth by the rhLf.

• Journal of Plant Biotechnology
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• v.32 no.3
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• pp.209-215
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• 2005

Lactoferrin is an iron-binding glycoprotein with many biological roles, including the protection against microbial and virus infection, stimulation of the immune system. We developed the transgenic Siberian ginseng (Acanthopanax senticosus) cell cultures producing the human lactoferrin (hLf) protein following Agrobacterium tumefaciens-mediated transformation. A construct containing a targeting signal peptide from tobacco endoplasmic reticulum fused to hLf cDNA under the control of an oxidative stress-inducible SWPA2 promoter was engineered. Transgenic Siberian ginseng cultured cells to produce a recombinant hLf protein were successfully generated and confirmed by PCR and Southern blot analysis. ELISA and western blot analysis showed that full length-hLf protein was synthesized in the transgenic cells. The production of hLf increased proportionally to cell growth and reached a maximal (up to 3% of total soluble proteins) at the stationary phase. These results suggest that the transgenic Siberian ginseng cultured cells in this study will be biotechnologically useful for the commercial production of medicinal plant cell cultures to produce hLf protein.

• Journal of Plant Biotechnology
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• v.37 no.4
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• pp.556-561
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• 2010

Lactoferrin is an 80-kDa iron-binding glycoprotein that is found in high concentrations in human milk. Human lactoferrin (hLF) has several beneficial biological activities including immune system modulation and antimicrobial activity. In the present study, we devolved a method of hLF expression through introducing the hLF gene construct into Oriza sativa cv. Nakdong using the Agrobacterium-mediated transformation system. The expression of the hLF gene under the control of the rice glutelin promoter was detected in the seeds of transgenic rice plants. Transformed rice plants were selected on media containing herbicide(DL-phosphinothricin) and the integration of hLF cDNA was confirmed by Southern blot analysis. The expression of the full length hLF protein from the grains of transgenic rice plants was verified by Western blot analysis. The lactoferrin expression levels in the transformed rice grains determined by enzyme-linked immunosorbant assay accounted for approximately 1.5% of total soluble protein. Taken together, these data indicate that rice grains expressing hLF can be directly incorporated into infant formula and baby food.

• Food Science of Animal Resources
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• v.25 no.1
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• pp.98-102
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• 2005

The purpose of this study was to demonstrate biochemical properties of lactoferrin (Lf) obtained from the colostrum of Korea native cow. The molecular weight of the purified Korean native cow's Lf (K-Lf) was 81kDa, the isoelectric point was 9, and the content of iron was 0.56 mg/g, which is indicated that iron saturation of the lactoferrin was 40.6%. Amino acid composition and a-helix content were different K-Lf from bovine Lf (B-Lf). Immunological cross reactivity was observed between K-Lf and B-Lf but not between K-Lf and human Lf (H-Lf) by immunodiffusion test and Western blot analysis. Out results indicate that structure of K-Lf is different from that of B-Lf although K-Lf and B-Lf were immunologically cross-reactive.