• Journal of Life Science
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• v.12 no.4
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• pp.496-503
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• 2002

To develop a yeast strain of Pichia pastoris producing an antibacterial peptide, we have attempted the expression and secretion of an insect defensin. The nucleotide sequences corresponding to mature defensin were chemically synthesized by 6 oligomers, assembled in vitro and the synthesized gene was identified by nucleotide sequencing. The prepro sequence of yeast mating factor $\alpha$1 and the defensin gene were recombined into a Pichia expression vector, pPIC9K. The resulting plasmid, pPIDE, was transformed into P. pastoris GSl15 and transformants selected on histidine-deficient minimal plates were tested for antibacterial activity against Micrococcus luteus. Four strains with different antibacterial activity were selected for further analysis. Southern hybridization and RT-PCR verified the defensin gene was maintained and transcribed in a host. Four strains were cultivated in YPD broth for 96 hours to compare cell growth and antibacterial activity, They showed no difference in cell growth, however, each strain showed different antibacterial activity pattern with culture time. The maximal activity was about 550 AU/ $m\ell$.

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.797-804
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• 2000

The human immunodeficiency virus type 1 (HIV-1) Tat is one of the viral gene products essential for HIV replication. The exogenous Tat protein is transduced through the plasma membrane and then accumulated in a cell. The basic domain of the Tat protein, which is rich in arginine and lysine residues and called the protein transduction domain (PTD), has been identified to be responsible for this transduction activity. To better understand the nature of the transduction mediated by this highly basic domain of HIV-1 Tat, the Green Fluorescent Protein (GFP) was expressed and purified as a fusion protein with a peptide derived from the HIV-1 Tat basic domain in Escherichia coli. The transduction of Tat-GFP into mammalian cells was then determined by a Western blot analysis and fluorescence microscopy. The cells treated with Tat-GFP exhibited dose- and time-dependent increases in their intracellular level of the protein. the effective transduction of denatured Tat-GFP into both the nucleus and the cytoplasm of mammalian cells was also demonstrated, thereby indicating that the unfolding of the transduced protein is required for efficient transduction. Accordingly, the availability of recombinant Tat-GFP can facilitate the simple and specific identification of the protein transduction mediated by the HIV-1 Tat basic domain in living cells either by fluorescence microscopy or by a fluorescence-activated cell sorter analysis.

• Journal of the Korean Magnetic Resonance Society
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• v.19 no.2
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• pp.74-82
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• 2015

Ryanodine receptor (RyR) is one of the two major $Ca^{2+}$ channels in membranes of intracellular $Ca^{2+}$ stores and is found in sarcoplasmic reticulum (SR), endoplasmic reticulum (ER). RyR1 is also the major calmodulin-binding protein of sarcoplasmic reticulum membranes. Residues 4064-4210 in the RyR1 polypeptide chain has similar primary sequence with calmodulin (CaM) and was designated as CaM-like domain (CaMLD). When expressed as a recombinant peptide, CaMLD showed several CaM-like properties in previous studies. Still, previous studies of CaMLD were focused on protein-protein interactions rather than its own properties. Here, we studied the expression of CaMLD and its sub-domains corresponding to each lobe of CaM in Escherichia coli. CaMLD could be obtained only as inclusion body, and it was refolded using urea solubilization followed by dialysis. Using spectroscopic approaches, such as NMR, circular dichroism, and gel filtration experiment, we found that the refolded CaMLD exists as nonspecific aggregate, even though it has alpha helical secondary structure. In comparison, the first half of CaMLD (R4061-4141) could be obtained as natively soluble protein with thioredoxin fusion. After the removal of the fusion tag, it exhibited folded and helical properties as shown by NMR and circular dichroism experiments. Its oligomeric status was different from CaMLD, existing as dimeric form in solution. However, the second half of the protein could not be obtained as soluble protein regardless of fusion tag. Based on these results, we believe that CaMLD, although similar to CaM in sequence, has quite different physicochemical properties and that the second half of the protein renders it the aggregative properties.

• BMB Reports
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• v.34 no.3
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• pp.201-205
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• 2001

Thionins are a family of low molecular weight cysteine-rich antimicrobial peptides. We isolated a cDNA encoding thionin gene from a flower bud cDNA library of Chinese cabbage (CFT). The gene contains 611 by nucleotides with 60 bp, and 150 by untranslated regions at its N- and C-terminal, respectively. The deduced amino acid sequence encoded 133 amino acids containing precursor polypeptide. The protein reveals that the precursor has a tripartite structure: a putative signal sequence at the N-terminus, followed by a mature thionin peptide, and a C-terminal acidic domain, which facilitates transport of the mature thionin through membrane. Genomic Southern blot analysis suggests that the CFT gene may be present as a single or two copy gene in the Chinese cabbage genome. Northern blot analysis shows that the gene is specifically expressed in flowers, but not in leaves, stems, or roots. When we analyzed the antifungal activity of the recombinant CFT protein, which was expressed in E. coli using the truncated cDNA region corresponding to the mature protein part, it was not active on fungal growth inhibition.

• Journal of Microbiology and Biotechnology
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• v.14 no.6
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• pp.1343-1349
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• 2004

A native strain of Pasteurella multocida was isolated from pigs suffering from severe atrophic rhinitis at domestic farms in Gyeonggi Province, Korea, and was identified as capsular serogroup 'D' and somatic serotype '4' by disc diffusion decapsulation and gel diffusion precipitation tests, respectively. The P. multocida (D:4) induced atrophic rhinitis in healthy pigs by the secondary infection. The gene for outer membrane protein H (ompH) of P. multocida (D:4) was cloned in Escherichia coli DH5$\alpha$ by PCR. The open reading frame of the ompH was composed of 1,023 bp, possibly encoding a protein with 341 amino acid residues containing a signal peptide of 20 amino acids at N-terminus, and the gene product with molecular mass of ca. 38 kDa was identified by SDS-PAGE. Hydropathy profiles indicated that there are two variable domains in the OmpH. To express the ompH in E. coli, the gene was manipulated in various ways. Expression of the truncated as well as full-length forms of the recombinant OmpH was fatal to the host E. coli BL21 (DE3). However, the truncated OmpH fused with GST was consecutively expressed in E. coli DH5$\alpha$. A large quantity of the fused polypeptide was purified through GST-affinity chromatography.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.257-264
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• 2009

A $\beta$-agarase gene, agaB34, was functionally cloned from the genomic DNA of a marine bacterium, Agarivorans albus YKW-34. The open reading frame of agaB34 consisted of 1,362 bp encoding 453 amino acids. The deduced amino acid sequence, consisting of a typical N-terminal signal peptide followed by a catalytic domain of glycoside hydrolase family 16 (GH-16) and a carbohydrate-binding module (CBM), showed 37-86% identity to those of agarases belonging to family GH-16. The recombinant enzyme (rAgaB34) with a molecular mass of 49 kDa was produced extracellularly using Escherichia coli $DH5{\alpha}$ as a host. The purified rAgaB34 was a $\beta$-agarase yielding neoagarotetraose (NA4) as the main product. It acted on neoagarohexaose to produce NA4 and neoagarobiose, but it could not further degrade NA4. The maximal activity of rAgaB34 was observed at $30^{\circ}C$ and pH 7.0. It was stable over pH 5.0-9.0 and at temperatures up to $50^{\circ}C$. Its specific activity and $k_{cat}/K_m$ value for agarose were 242 U/mg and $1.7{\times}10^6/sM$, respectively. The activity of rAgaB34 was not affected by metal ions commonly existing in seawater. It was resistant to chelating reagents (EDTA, EGTA), reducing reagents (DTT, $\beta$-mercaptoethanol), and denaturing reagents (SDS and urea). The E. coli cell harboring the pUC18-derived agarase expression vector was able to efficiently excrete agarase into the culture medium. Hence, this expression system might be used to express secretory proteins.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.47 no.4
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• pp.370-375
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• 2014

BACE2 is a membrane-bound aspartic protease that is highly homologous with BACE1. While BACE1 processes the amyloid precursor protein (APP) at a key step in generating ${\beta}$-amyloid peptide and presumably causes Alzheimer's disease (AD), BACE2 has not been demonstrated to be involved in APP processing directly, and its physiological functions are unknown. To determine its function and to develop inhibitors from marine sources, we constructed an overexpression vector for producing BACE2. The gene encoding human BACE2 protease was amplified using the polymerase chain reaction and cloned into the pET11a expression vector, resulting in pET11a/BACE2. Recombinant BACE2 protease was overexpressed successfully in E. coli as inclusion bodies, refolded using the rapid-dilution method, and purified via two-step fast protein liquid chromatography using Sephacryl S-300 gel filtration and Resource-Q column chromatography. The BACE2 protease produced was an active form. This study provides an efficient method not only for studying the basic properties of BACE2, but also for developing inhibitors from natural marine sources.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.6
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• pp.850-855
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• 2007

Leptin is the adipocyte-specific product of the obese gene and plays a major role in food intake and energy metabolism. Leptin research was mainly focused on mammalian species, but understanding of leptin and its function in poultry is very poor. In this study, the duck leptin gene was amplified using the reverse transcription-polymerase chain reaction (RT-PCR) from duck liver RNA. The cDNA fragment was inserted into the pET-28a expression vector, and the resulting plasmid was expressed in Escherichia coli BL21 (DE3). Experimental mice were given an intraperitoneal injection of 10 mg/kg leptin dissolved in phosphate buffered saline (PBS), while the control mice were injected with PBS. The effect of leptin on food intake, body weight and fatty deposition in mice was detected. Sequence analysis revealed that duck leptin had a length of 438 nucleotides which encoded a peptide with 146 amino acid residues. The sequence shares highly homology to other animals. The coding sequence of duck leptin was 84 and 86% identical to human and pig leptin nucleotides sequence. Highest identity was with the rat coding sequence (95%). The identity of the amino acid sequence was 84, 82 and 96% respectively compared to that of the human, pig and rat. Results of SDS-PAGE analysis indicated that a fusion protein was specifically expressed in E. coli BL21 (DE3). The purified product was found to be biologically active during tests. Continuous administration of recombinant duck leptin inhibited food intake. Despite the decrease of food intake, leptin significantly induced body weight and fatty deposition. These changes were accompanied by a significant down-secretion of plasma glucose, cholesterol, triglyceride and insulin levels in mice. The observations provide evidence for an inhibitory effect of leptin in the regulation of food intake and for a potential role of duck leptin in the regulation of lipogenesis.

• Journal of Microbiology and Biotechnology
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• v.19 no.2
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• pp.194-203
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• 2009

$HpaG_{Xooc}$, from rice pathogenic bacterium Xanthomonas oryzae pv. oryzicola, is a member of the harpin group of proteins, eliciting hypersensitive cell death in non-host plants, inducing disease and insect resistance in plants, and enhancing plant growth. To express and secret the $HpaG_{Xooc}$ protein in Bacillus subtilis, we constructed a recombinant expression vector pM43HF with stronger promoter P43 and signal peptide element nprB. The SDS-PAGE and Western blot analysis demonstrated the expression of the protein $HpaG_{Xooc}$ in B. subtilis. The ELISA analysis determined the optimum condition for $HpaG_{Xooc}$ expression in B. subtilis WBHF. The biological function analysis indicated that the protein $HpaG_{Xooc}$ from B. subtilis WBHF elicits hypersensitive response(HR) and enhances the growth of tobacco. The results of RT-PCR analysis revealed that $HpaG_{Xooc}$ induces expression of the pathogenesis-related genes PR-1a and PR-1b in plant defense response.

• Animal cells and systems
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• v.11 no.2
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• pp.175-182
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• 2007

The lysozyme II gene of cabbage butterfly Artogeia rapae was cloned from fat body of the larvae injected with E. coli and its nucleotide sequence was determined by the RACE-PCR. It has an open reading frame of 414 bp nucleotides corresponding to 138 amino acids including a signal sequence of 18 amino acids. The estimated molecular weight and the isoelectric point of the lysozyme II without the signal peptide were 13,649.38 Da and 9.11, respectively. The A. rapae lysozyme II (ARL II) showed the highest identity (81%) in the amino acid sequence to Manduca sexta lysozyme among other lepidopteran species. The two catalytic residues ($Glu^{32}$ and $Asp^{50}$) and the eight Cys residue motifs, which are highly conserved among other c-type lysozymes in invertebrates and vertebrates, are also completely conserved. A phylogenetic analysis based on amino acid sequences indicated that the ARL II was more closely related to M. sexta, Hyphantria cunea, Heliothis virescens, and Trichoplusia ni lysozymes. The ARL II gene was expressed in Spodoptera frugiperda 21 insect cells and the recombinant ARL II (rARL II) was purified from cell-conditioned media by cation exchange column chromatography and reverse phase FPLC. The purified rARL II was able to form a clear zone in lysoplate assay against Micrococcus luteus. The lytic activity was estimated to be 511.41 U/mg, 1.53 times higher than that of the chicken lysozyme. The optimum temperature for the lytic activity of the rARL II was $50^{\circ}C$, the temperature dependency of the absolute lytic activity of rARL II was higher than that of the chicken lysozyme at low temperatures under $65^{\circ}C$.