• Journal of Microbiology and Biotechnology
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• v.21 no.7
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• pp.667-674
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• 2011

The gene for esterase (rEst1) was isolated from a new species of genus Rheinheimera by functional screening of E. coli cells transformed with the pSMART/HaeIII genomic library. E. coli cells harboring the esterase gene insert could grow and produce clear halo zones on tributyrin agar. The rEst1 ORF consisted of 1,029 bp, corresponding to 342 amino acid residues with a molecular mass of 37 kDa. The signal P program 3.0 revealed the presence of a signal peptide of 25 amino acids. Esterase activity, however, was associated with a homotrimeric form of molecular mass 95 kDa and not with the monomeric form. The deduced amino acid sequence showed only 54% sequence identity with the closest lipase from Cellvibrio japonicus strain Ueda 107. Conserved domain search and multiple sequence alignment revealed the presence of an esterase/ lipase conserved domain consisting of a GXSXG motif, HGGG motif (oxyanion hole) and HGF motif, typical of the class IV hormone sensitive lipase family. On the basis of the sequence comparison with known esterases/ lipases, REst1 represents a new esterase belonging to the class IV family. The purified enzyme worked optimally at $50^{\circ}C$ and pH 8, utilized pNP esters of short chain lengths, and showed best catalytic activity with p-nitrophenyl butyrate ($C_4$), indicating that it was an esterase. The enzyme was completely inhibited by PMSF and DEPC and showed moderate organotolerance.

• Microbiology and Biotechnology Letters
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• v.49 no.2
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• pp.148-156
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• 2021

Single-chain antibodies against epidermal growth factor receptor variant III (EGFRvIII) are potentially promising agents for developing antibody-based cancer treatment strategies. We described in our previous study the successful expression of an anti-EGFRvIII scFv antibody in Escherichia coli. However, we could also observe the formation of insoluble aggregates in the periplasmic space, limiting the production yield of the active product. In the present study, we investigated the mechanisms by which growth conditions could affect the expression of the soluble anti-EGFRvIII scFv antibody in small-scale E. coli NiCo21(DE3) cultures, attempting to maximize production. The secreted scFv molecules were purified using Ni-NTA magnetic beads and protein characterization was performed using SDS-PAGE and western blot analyses. We used the ImageJ software for protein quantification and determined the antigen-binding activity of the scFv antibody against the EGFRvIII protein. Our results showed that the highest percentage of soluble scFv expression could be achieved under culture conditions that combined low IPTG concentration (0.1 mM), low growth temperature (18℃), and large culture dish surface area. We found moderate-yield soluble scFv production in the culture medium after lactose-mediated induction, which was also beneficial for downstream protein processing. These findings were confirmed by conducting western blot analysis, indicating that the soluble, approximately 30-kDa scFv molecule was localized in the periplasm and the extracellular space. Moreover, the antigen-binding assay confirmed the scFv affinity against the EGFRvIII antigen. In conclusion, our study reveals that low-speed protein expression is preferable to obtain more soluble anti-EGFRvIII scFv protein in an E. coli expression system.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.56 no.1
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• pp.21-27
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• 2023

Lotus Nelumbo nucifera seed protein (LSP) was isolated by alkaline solubilization after removing fat and phenolics by hexane and ethanol treatment. Antioxidant peptides from LSP were produced with Alcalase^® and pepsin and hydroxyl radical scavenging activities were determined. LSP-Alcalase^® hydrolysates showed higher hydroxyl radical scavenging activity than LSP-pepsin hydrolysates. To purify antioxidant peptides, LSP-Alcalase^® hydrolysates were subjected to high performance liquid chromatography (HPLC) separation on the C18 column and the active fraction was further purified using a Superdex^TM peptide 10/300 GL column. Finally, the active fraction (F8-2) was evaluated for antioxidant activities by 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radical scavenging, and oxygen radical absorbance capacity (ORAC) assays. The EC₅₀ values of the F8-2 were 105.81±0.02 ㎍/mL for DPPH and 32.26±0.02 ㎍/mL for hydroxyl radical and the F8-2 exhibited 7.22 μM trolox equivalent (TE)/100 ㎍ F8-2. Glutathione (GSH), which is a positive control, showed EC₅₀ values of 19.87±0.01 ㎍/mL for DPPH and 15.95±0.03 ㎍/mL for hydroxyl radical and an ORAC value of 14.17±0.03 μM TE/100 ㎍ GSH. Finally, sixteen peptides were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among them, Ile-Tyr and Leu-Tyr showed higher antioxidant scores.

• Journal of the Korean Society of Food Science and Nutrition
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• v.40 no.6
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• pp.903-911
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• 2011

In this study, Bacillus polyfermenticus CJ6 harboring antibacterial activity was isolated from meju. The antibacterial activity of Bacillus polyfermenticus CJ6 was stable in the pH range of 3.0~9.0, but it disappeared after culture at $70^{\circ}C$ for 24 hr. Antibacterial activity was inactivated by proteinase K, protease, and ${\alpha}$-chymotrypsin, indicating its proteinaceous nature. The growth inhibitory effects of B. polyfermenticus CJ6 culture on food-borne pathogens such as Staphylococcus aureus, Salmonella Typhi, Listeria monocytogenes, and Escherichia coli O157:H7 were examined in this study. Approximately 6~6.2 log CFU/mL of each pathogen was co-cultured with B. polyfermenticus CJ6 in a 50 mL culture volume for 24 hr. Growth of S. aureus and L. monocytogenes was completely inhibited after 3 hr of incubation. Growth of S. Typhi and E. coli O157:H7 was also completely inhibited after 6 hr of incubation. The antibacterial compounds from B. polyfermenticus CJ6 were purified by solid phase extraction (C18 Sep-pak cartridge), recycling preparative HPLC, and analytical HPLC. Ultra-high performance liquid chromatography and electrospray ionization tandem mass spectrometry analysis were used to identify the purified antibacterial compounds, which were confirmed to be five peptides (757.4153 Da, 750.3444 Da, 1024.5282 Da, 1123.6083 Da, and 1617.8170 Da).

• The Korean Journal of Mycology
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• v.40 no.4
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• pp.229-234
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• 2012

A key molecule in the pathogenesis of Alzheimer's disease (AD) is the ${\beta}$-amyloid peptide ($A{\beta}$) generated by ${\beta}$-secretase activity, an aspartic protease. This study was designed to evaluate inhibitory effect of the high-molecular weight water-soluble polysaccharides (Et-P) isolated and purified from Phellinus linteus fruiting body on ${\beta}$-secretase activity. The Et-P was purified from the hot water extract of Phellinus linteus fruiting body mainly by 75% ethanol precipitation and DEAE-Cellulose column chromatography. From the DEAE-Cellulose chromato-gram and molecular weight analysis, the Et-P was shown to be a mixture of three polysaccharides with molecular mass of 1,629, 1,294, and 21 kDa, respectively. The monosaccharide composition of Et-P was determined to be glu-cose, galactose, and mannose as major sugars, glucose being the most prominent one (48% in mole percentage). The elemental analysis and FT-IR analysis suggested that Et-P is typical polysaccharides having at least partially ${\beta}$-linkages and possible existing as complex with phenolic compounds. The laminarinase digestion and HPAEC-PAD analysis suggested that Et-P is a variant of beta-(1,3)-glucans. The Et-P showed DPPH radical scavenging activity and, especially, a significant inhibitory activity on ${\beta}$-secreatase activity (48% inhibitin at 100 ${\mu}g/mL$), suggesting that they may inhibit the formation of $A{\beta}$ which is the major causative of Alzheimer's disease. The results of this study suggest that the water soluble polysaccharides of Phellinus linteus fruiting body can be a potent material for the development of preventive or therapeutic agents for AD.

• Korean Journal of Agricultural Science
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• v.25 no.1
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• pp.52-67
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• 1998

The investigative research on the mammalian milk purely consisted of the physiological quality of lactoferrin was conducted to reveal the antimicrobial ativity of specifically functional foods with antibiotic characteristics as a basic data in food manufacturing. Bovine lactoferrin were isolated from raw milk samples, and was digested with pepsin, trypsin and chymotrypsin. It was necessary then to separate and purify lactoferrin from bovine raw milk, and in order to analyze the antimicrobial activity of the enzyme-treated bovine lactoferrin in their required quantitative fraction. Afterwards Escherichia coli and Staphylococcus aureus was incubated in it. It was that investigated to enzyme-treated fractions molecular weight and the peptide fragment with antimicrobial effect. 1. The purity of enzyme-treated bovine lactoferrin(BLF) was tested by SDS-PAGE. As a results of 12% SDS-PAGE assay, pepsin-treated LF did not exhibited band until if reaches 14 KDa, while trypsin and chymotrypsin treated LF, known to contain the non-digestive lactoferrin exhibited band at a molecular weight of 33 KDa. 2. Bovine lactoferrin was sucessfully purified through the use of Sephadex G-50 Column. In order to assay LF through the Sephadex G-50 column chromatography, the digestive bovine lactoferrin (BLFs) was eluted with a linear gradient of 0.05% Tris-HCl. When the gel-filtration analysis, pepsin, trypsin and chymotrypsin treatments of BLF fragments was showed 2, 3, and 2 peak, respectively. The results of the HPLC analysis confirmed that had a non-digestive lactoferrin receptor, and trypsin and chymotrypsin treated BLFs has an antimicrobial effect. 3. To measure the strength of the antimicrobial effect of enzyme treated lactoferrin it was compared to the antimicrobial activity taking place at the incubated Escherichia coli and Staphylococcus aureus. This might explain the resistance of the microorganisms for peptide fragment. The pepsin-treated of bovine lactoferrin was markedly reduced by incubation of the cells. Trypsin-treated of BLF was similar to chymotrypsin-treated of BLF. However, trypsin and chymotrypsin treatments of BLFs were showed the antimicrobial effect until eight hours incubation for native bovine lactoferrin. Therefore the enzyme-treated lactoferrin have an antimicrobial effect even non-digestive lactoferrin. 4. The digestive bovine lactoferrin fragments assay was carried out by the use of Sephadex G-50 column chromatography and SDS-PAGE. The pepsin and chymotrypsin-treated fragments has a low molecular weight and trypsin-treated lactoferrin was only showed a band. It was described that characteristics of digestive protein. It appeared that there may be a relation between virulence and resistance to enzyme-treated BLF.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.45 no.2
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• pp.175-184
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• 2019

The epidermal growth factor (EGF) has a intrinsic function of inducing growth and proliferation of cells through interacting with cell membrane receptors in human epidermis and dermis layer. These functions of EGF are used as a main ingredient for wound healing medicines and anti-aging cosmetics. As a cosmetic ingredient, the EGF has a problem in exhibiting its natural efficacy due to the lack of the ability to penetrate through the stratum corneum, which is known as the skin barrier. In this study, a recombinant human epidermal growth factor ($MTD_{151}-EGF$) fused with the macromolecule transduction domain $(MTD)_{151}$ with the skin penetration ability was developed to improve the skin penetration efficiency of the EGF. Expression of $MTD_{151}-EGF$ was performed in E. coli transformed with a vector encoding the $MTD_{151}-EGF$ gene and then purified. The purified $MTD_{151}-EGF$ was evaluated using cell proliferation assay, cytotoxicity test and skin penetration test by franz diffusion cell assay and artificial skin. Cell proliferation activity of $MTD_{151}-EGF$ purified to high purity of 99% or above was equivalent to the EGF or better, and cytotoxicity was not observed. In addition, the $MTD_{151}-EGF$ showed an excellent penetration efficiency compared to the EGF in the skin penetration test with EGF and $MTD_{151}-EGF$ labeled by FITC in an artificial skin penetration model. Based on the quantitative analysis of the penetrating substance using franz diffusion cell assay, the amount of penetration was about 16 times more than that of EGF. These results can be regarded as an effective alternative to improve the existing physical transdermal penetration method related to the use of various active ingredients for cosmetics.

• Journal of Microbiology
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• v.44 no.5
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• pp.548-555
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• 2006

FaeG is the key factor in the infection process of K88ad enterotoxigenic Escherichia coli (ETEC) fimbrial adhesin. In an attempt to determine the possibility of expressing recombinant FaeG with immunogenicity for a new safe and high-production vaccine in E. coli, we constructed the recombinant strain, BL21 (DE3+K88), which harbors an expression vector with a DNA fragment of faeG, without a signal peptide. Results of 15% SDS-polyacrylamide slab gel analysis showed that FaeG can be stably over-expressed in BL21 (DE3+K88) as inclusion bodies without FaeE. Immunoglobulin G (IgG) and M (IgM) responses in pregnant pigs, with boost injections of the purified recombinant FaeG, were detected 4 weeks later in the sera and colostrum. An in vitro villius-adhesion assay verified that the elicited antibodies in the sera of vaccinated pigs were capable of preventing the adhesion of K88ad ETEC to porcine intestinal receptors. The protective effect on the mortality rates of suckling piglets born to vaccinated mothers was also observed one week after oral challenge with the virulent ETEC strain, $C_{83907}$ (K88ad, $CT^+,\;ST^+$). The results of this study proved that the adhesin of proteinaceous bacterial fimbriae or pili could be overexpressed in engineered E. coli strains, with protective immune responses to the pathogen.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.33 no.1
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• pp.55-59
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• 2000

A specific and sensitive sandwich enzyme-immunoassay (EIA) using Avidin-Biotin complex was developed for the measurement of GTH II levels in pituitary content and pituitary cell culture medium of the rainbow trout-(Oncorhpchus mykiss). Biotin-salmon GTH II rabbit IgG (sefondary antibody) wai purified by a protein A sepharose affinity chromatography column and that was biotinylated by using Biotin-N-hydroxysuccinimide ofter (BNHS). Non-biotin salmon GTH II rabbit IgG (first antibody) was obtained only through a protein A sepharose affinity chromatography column. The assay was performed by the so-called 'sandwich' method using a microtiter plate, A dose-response curve was obtained between $0.12 to 125 ng/ml$ of salmon GTH II. The displacement curves for pituitary extraction and pituitary cell culture medium of testosterone-treated rainbow trout were Parallel to the standard curie. The intra-assay and inter-assay coefficients of variation (CV) were $8.2{\%} (N=5) and 12.5{\%} (N=6)$, respectively, This assay system was used to measure the amount of GTH II that accumulated in the culture medium of dispersed pituitary cells in testosterone-treated immature rainbow trout, The accumulation was increased with the amount or salmon gonadotropin-releasing hormone. GTH II values determined by the present method were well correlated with those determined by radioimmunoassay. As a result, this assay system was found to be suitable for the measurement of GTH II for pituitary extraction and pituitary culture medium in many salmonid fishes.

• Korean Journal of Poultry Science
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• v.38 no.4
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• pp.323-330
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• 2011

The chicken monoclonal antibody (mAb), 13C8, reacts with sporozoite antigens of Eimeria spp. which causes avian coccidiosis. Since this mAb was produced at low amount due to genetic instability of chicken hybridoma, a recombinant 13C8 single-chain Fv (ScFv) antibody was constructed by amplification of the variable domain of heavy (VH) and light chain (VL) genes of antibody derived from chicken hybridoma. The constructed 13C8 ScFv was successfully expressed in E. coli and purified as a soluble form. In ELISA analysis, this recombinant 13C8 ScFv antibody showed antigen binding activity as the original mAb. In addition, nucleotide sequence comparison of 13C8 gene to the germline chicken VL and VH genes suggested that the gene conversion with $V{\lambda}$ and VH pseudogenes might contribute to the diversification of VL and VH genes in chickens.