• BMB Reports
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• v.41 no.5
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• pp.408-413
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• 2008

Pyridoxal-5'-phosphate phosphatase (PLPP) catalyzes the dephosphorylation of pyridoxal-5'-phosphate (PLP). A human brain PLPP gene was fused with a PEP-1 peptide and produced a genetic in-frame PEP-1-PLPP fusion protein. The purified PEP-1-PLPP fusion protein was efficiently transduced into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced PEP-1-PLPP fusion protein was stable for 36 h. The concentration of PLP was markedly decreased by the addition of exogenous PEP-1-PLPP to media pretreated with the vitamin $B_6$ precursors; pyridoxine, pyridoxal kinase and pyridoxine-5'-phosphate oxidase into cells. The results suggest that the transduction of the PEP-1-PLPP fusion protein can be one mode of PLP level regulation, and to replenish this enzyme in the various neurological disorders related to vitamin $B_6$.

• BMB Reports
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• v.42 no.3
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• pp.136-141
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• 2009

Familial Amyotrophic lateral sclerosis (FALS) is a progressive neurodegenetative disorder induced by mutations of the SOD1 gene. Heat shock protein 27 (HSP27) is well-defined as a stress-inducible protein, however the its role in ALS protection has not yet been established. To investigate the role HSP27 may have in SOD1 mutant-mediated apoptosis, human SOD1 or HSP27 genes were fused with a PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame fusion protein, which was then transduced into cells. We found the purified PEP-1-HSP27 fusion proteins can be transduced efficiently into neuronal cells and protect against cell death by enhancing mutant SOD1 activity. Moreover, transduced PEP-1-HSP27 efficiently prevents protein aggregation produced by oxidative stress. These results suggest that transduced HSP27 fusion protein may be explored as a potential therapeutic agent for FALS patients.

• The Korean Journal of Mycology
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• v.27 no.4 s.91
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• pp.293-298
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• 1999

An one percent sodium carbonate extract prepared from sclerotia of Poria cocos activated the proliferation of the T lymphocytes as measured by mixed lymphocyte responses(MLR). The active fraction, PCSC22, was isolated from an one percent sodium carbonate extract by a combination of fractionation procedures, including ethanol precipitation and chromatographies on column of DEAE-cellulose and Sephadex G50. Carbohydrate and peptide contained in PCSC22 were 78 : 22% in ratio. On employing gel filtration high performance liquid chromatography, PCSC22 exhitited a homogeneous peak with an average molecular weight of 8 kDa. The sugar moiety of PCSC22 was composed with mannose (92%), galactose (6.2%) and arabinose (1.3%), which might be indicated as heteromannan. Fifteen amino acids were found in peptide moiety of the polysaccharide and aspartic acid, serine, and valine were major components. PCSC22 activated the primary proliferation of T lymphocytes measured by mixed lymphocyte responses, the antibody production of the B lymphocytes and the secretion of nitric oxide from macrophage cell line, RAW264.7.

• Journal of Life Science
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• v.25 no.6
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• pp.715-723
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• 2015

Insects represent the largest class within the animal kingdom in terms of species number. Humans had been utilized insect in the broad area, including food, agriculture, industry, pharmaceuticals and so on. At present, insects are emerging as a leading group for identifying and extracting novel bioactive substances due to enormous number and a high nutritional value. Insects rely on a suite of systemic response to resist infection such as immune cells, hemocytes, activation of enzymes cascades, and antimicrobial peptide/protein. Among the substances, antimicrobial peptides (AMPs) are main components of potent antimircrobial innate defense system into the insect hemolymph. AMPs raise influential candidate as avenue to resolve the development of antibiotic-resistant microbial organism. Insect AMPs are classified into four main classes: cecropins, insect defensins, glycine/proline-rich peptides. Insect AMPs have been purified, over 150. In this review, AMPs derived from several insects were summarized including honey bee, dung beetle, butterfly and longicorn beetle. These peptides almost exhibited potent antimicrobial activities against human microbial pathogens without causing remarkable hemolysis to erythrocytes excluding melittin, and their mode of action(s) are based on disruption of the plasma membrane or fungal apoptosis. Therefore, study of insect AMPs is expected to be useful for designing novel therapeutic antimicrobial applications.

• BMB Reports
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• v.41 no.7
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• pp.537-541
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• 2008

Epilepsy is characterized by the presence of spontaneous episodes of abnormal neuronal discharges and its pathogenic mechanisms remain poorly understood. Recently, we found that the expression of creatine kinase (CK) was markedly decreased in an epilepsy animal model using proteomic analysis. A human CK gene was fused with a HIV-1 Tat peptide to generate an in-frame Tat-CK fusion protein. The purified Tat-CK fusion protein was efficiently transduced into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced Tat-CK fusion protein was stable for 48 h. Moreover, the Tat-CK fusion protein markedly increased endogenous CK activity levels within the cells. These results suggest that Tat-CK provides a strategy for the therapeutic delivery of proteins in various human diseases including the delivery of CK for potential epilepsy treatment.

• Journal of Applied Biological Chemistry
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• v.61 no.4
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• pp.405-410
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• 2018

Pseudomonas tolaasii strain No. 6264 has been isolated from mushroom tissue and identified as one of the major pathogen causing brown blotch disease. It secretes peptide toxins, known as tolaasin and its analogue peptides. P. tolaasii 6264 has been used as a typical pathogenic strain to study the brown blotch disease for last 20 years after confirming its blotch-forming ability, hemolytic activity, and white line formation. In this study, the characteristics of P. tolaasii 6264 strain were analyzed and compared according to storage period. Strains of P. tolaasii 6264 stored annually since 2012 were cultured and their pathogenic characters were analyzed. When the 16S rRNA sequences were compared, all strains were divided into two groups. Pathogenic characters including hemolytic activity, blotch-forming ability, and white line test were also investigated. The strains, P. tolaasii 6264-15-2 and P. tolaasii 6264-17, had all three activities; however, the rest of stored strains showed only blotch-forming ability losing other pathogenic characters. Tolaasin peptides were purified from the bacterial cultures and analyzed by mass spectrometry. The strains, P. tolaasii 6264-15-2 and P. tolaasii 6264-17, secreted Tol I (1987 Da), Tol II (1943 Da), and its analogues (1973 Da, 2005 Da) while some of these peptides were not found in the media cultured other strains. These results indicate that the pathogenicity of P. tolaasii could be varied during the storage period.

• Journal of Mushroom
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• v.19 no.4
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• pp.285-293
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• 2021

The purpose of this study was to identify and characterize the laccase genes of Flammulina velutipes var. lupinicola. Five laccase genes (g1934, g1937, g2415, g2539, g5858) were selected based on the copper binding site and signal peptide analysis results using the laccase gene selected from the F. velutipes var. lupinicola genome. The size of the laccase genes of F. velutipes var. lupinicola were 1,488 bp~1,662 bp. As a result of cDNA sequence analysis, 14 to 17 introns were identified in the laccase genes. The cleavage site predicted as the signal peptide of the laccase gene was found to be located between 20 bp and 34 bp from the N-terminus. In addition, separation and purification were performed to characterize the F. velutipes var. lupinicola laccases, and the optimal activity of the separated and purified proteins were analyzed by pH, temperature and time. Five bands with laccase activity were found from zymogram analysis. The optimal pH of the reaction was 5.5, the optimal temperature was found to be 40℃. Therefore, characterization of the laccase genes identified in this study should help in better understanding the biomass decomposition of F. velutipes var. lupinicola.

• 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.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.999-1006
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• 2015

The endo-polygalacturonase gene (endo-pgaA) was cloned from DNA of Aspergillus niger SC323 using the cDNA synthesized by overlapping PCR, and successfully expressed in Saccharomyces cerevisiae EBY100 through fusing the α-factor signal peptide of yeast. The fulllength cDNA consists of 1,113 bp and encodes a protein of 370 amino acids with a calculated molecular mass of 38.8 kDa. After induction by galactose for 48 h, the activity of recombinant endo-PgaA in the culture supernatant can reach up to 1,448.48 U/mg. The recombinant protein was purified to homogeneity by ammonium sulfate precipitation and gel filtration column chromatography and subsequently characterized. The optimal pH and temperature of the purified recombinant enzyme were 5.0 and 50℃, respectively. The Michaelis-Menten constant (K_m) and maximal velocity (V_max) of the enzyme for pectin were 88.54 μmol/ml and 175.44 μmol/mg/min, respectively. The enzyme activity was enhanced by Ca²⁺, Cu²⁺, and Na⁺, and strongly inhibited by Pb²⁺ and Mn²⁺. The pectin hydrolysates were mainly galacturonic acid and other oligo-galacturonates. Therefore, these characteristics suggest that the recombinant endo-PgaA may be of potential use in the food and feed industries.

• The Korean Journal of Physiology
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• v.23 no.1
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• pp.89-98
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• 1989

In order to produce antibody for use in radioimmunoassay of gastrin in physiological concentration, four rabbits of New Zealand white were immunized with synthetic human gastrin-17-I conjugated to hemocyanin with EDC. Among them, only one rabbit produced antibody that could bind 50% of $^{125}I-gastrin$ at a final dilution of 1:25,000. $^{125}I-gastrin$ was prepared with synthetic human gastrin-17-I and $NaI^{125}$ by lactoperoxidase technique. The product was then purified on a column of Sephadex Gl5/G5O (7:3, w/w) followed by a column of DEAE sephadex A-25. The specific radioactivity of the purified $^{125}I-gastrin$ was in the range of 347-1429 ${\mu}Ci/nmole$ when determined by the self-displacement method. The effective affinity constant $(K_{eff})$, total binding sites (N), heterogeneity index $({\alpha})$ and average affinity constant $(K_{0})$ of the anti-gastrin serum calculated from Scatchard plot as well as Sips plot were $1.77{\times}10^{11}/M$, 255 nM, 0.84 and $0.79{\times}10^{11}/M$, respectively. When radioimmunoassay was performed with the anti-gastrin serum, it was confirmed that the mean concentration of gastrin immunoreactivity in plasma was increased by feeding in humans and rats, and also increased by bombesin administration in rats. The results indicate that the anti-gastrin serum produced in the present investigation is suitable for radioimmunological determination of gastrin in physiological concentration.