• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.4
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• pp.304-308
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• 2005

The response of IPTG induction was investigated through the monitoring of the alkali consumption rate and buffer capacity during the cultivation of recombinant E. coli BL21 (DE3) harboring the plasmid pRSET-LacZ under the control of lac promoter. The rate of alkali consumption increased along with cell growth, but declined suddenly after approximately 0.2 h of IPTG induction. The buffer capacity also declined after 0.9 h of IPTG induction. The profile of buffer capacity seems to correlate with the level of acetate production. The IPTG response was monitored only when introduced into the mid-exponential phase of bacterial cell growth. The minimum concentration of IPTG for induction, which was found out to be 0.1 mM, can also be monitored on-line and in-situ. Therefore, the on-line monitoring of alkali consumption rate and buffer capacity can be an indicator of the metabolic shift initiated by IPTG supplement, as well as for the physiological state of cell growth.

• Korean journal of applied entomology
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• v.53 no.4
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• pp.331-338
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• 2014

Cystatins (CSTs) are reversible and competitive inhibitors of C1A cysteine proteases, corresponding to papain-like cathepsins in plants and animals. A viral CST (CpBV-CST1) was identified from a polydnavirus, Cotesia plutellae bracovirus (CpBV). Our previous study indicated that a transient expression of CpBV-CST1 interfered with immune response and development of Plutella xylostella larvae. To directly demonstrate the protein function, this study produced a recombinant CpBV-CST1 protein (rCpBV-CST1) using bacterial expression system to determine its inhibitory activity against cysteine protease and to assess its physiological alteration in insect immune and development. The open reading frame of CpBV-CST1 encodes a polypeptide of 138 amino acids (${\approx}15kDa$). rCpBV-cystatin protein in BL21 STAR (DE3) competent cells containing a recombinant pGEX4T-3:CpBV-CST1 was over-expressed by 0.5 mM IPTG for 4 h. In biological activity assay, the purified rCpBV-CST1 showed a significant inhibition against papain activity. It inhibited a cellular immune response of hemocyte nodule formation in the beet armyworm, Spodoptera exigua. Moreover, its oral administration retarded larval development of the diamondback moth, Plutella xylostella in a dose-dependent manner. These results suggest that CpBV-CST1 may be applied to control insect pest populations.

• Biomedical Science Letters
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• v.12 no.3
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• pp.139-143
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• 2006

Jopock (Jpk) has previously been ascertained that induces both bacterial and mammalian cell death. The Escherichia coli cells expressing Glutathion S-transferase (GST) fused Jpk showed elongated phenotype and inhibited cell growth which led eventual cell death. In an attempt to search the genetic suppressor of the lethal protein Jpk in bacterial cells, we constructed a unidirectional protein expression vector inserting tac promoter next to the C-terminus Jpk in pGEX-Jpk. The function of additional tac promoter was confirmed by substituting lac promoter in Plac-TOPO plasmid. The cells harboring plac- TOPO, which regulates $lacZ{\alpha}$ gene expression under lac promoter, formed blue colonies in 5-bromo-4-3 $indolyo-{\beta}-D-galactoside$ (X-gal) plate. When lac promoter was changed to tac promoter, same results were observed. Since the addition of tac promoter did not affect the toxic effect of Jpk, the pGEX-Jpk-ptac could be a useful vector for the screening of suppressor(s) for Jpk, in which GST-Jpk and a putative Jpk-suppressing protein are coexpressing from two unidirectional tac promoters, which response to the same inducer, $isopropyl-{\beta}-D-thiogalactopyranoside (IPTG)$.

• Animal cells and systems
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• v.16 no.6
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• pp.455-461
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• 2012

Amongst the various antimicrobial peptides, lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. Here we propose the biosynthesis and refolding of recombinant lysozyme in Escherichia coli expressed in inclusion body form. The Agrius lysozyme gene was amplified using gene specific primers and then ligated into the pGEX-4T-1 vector, which contained the glutathione S-transferase (GST) gene as a fusion partner. A recombinant lysozyme was expressed in E. coli Rosetta cells using a pGEX-4T-1 expression vector, and the fusion protein was induced by ioporpyl-${\beta}$-D-thiogalactopyranoside (IPTG). The recombinant protein produced as an inclusion body was resolubilized in solubilization buffer, and the resultant solution was dialyzed in refolding buffer. After thrombin cleavage, the recombinant lysozyme was purified by ion exchange chromatography and reverse phase chromatography. The recombinant lysozyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoreactivity against the anti-Agrius lysozyme was observed by western blot analysis of this protein. The recombinant lysozyme displayed antibacterial activity against Bacillus megaterium and Micrococcus luteus, which was confirmed by the inhibition zone assay.

• Journal of Microbiology and Biotechnology
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• v.22 no.4
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• pp.510-515
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• 2012

A recombinant putative dextransucrase (DexT) was produced from Leuconostoc citreum KM20 as a 160 kDa protein, but its productivity was very low (264 U/l). For optimization, we examined enzyme activity in 7 Escherichia coli strains with inducer molecules such as lactose or IPTG. E. coli BL21-CodonPlus(DE3)-RIL exhibited the highest enzyme activity with lactose. Finally, DexT activity was remarkably increased by 12-fold under the optimized culture conditions of a cell density to start induction ($OD_{600}$) of 0.95, a lactose concentration of 7.5 mM, and an induction temperature of $17^{\circ}C$. These results may effectively apply to the heterologous expression of other large DexT genes.

• Journal of Plant Biotechnology
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• v.43 no.1
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• pp.30-36
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• 2016

Brassinosteroids (BRs) are essential plant steroid hormones required for cell elongation, plant growth, development and abiotic and biotic stress tolerance. BRs are recognized by BRI1 receptor kinase that is localized in the plasma membrane, and the BRI1 protein will eventually autophosphorylate in the intracellular domain and transphosphorylate BAK1, which is a co-receptor in Arabidopsis thaliana. However, little is known of the role OsBRI1 receptor kinase plays in Oryza sativa, monocotyledonous plants, compared to that in Arabidopsis thaliana, dicotyledonous plants. As such, we have studied OsBRI1 receptor kinase in vitro and in vivo with recombinant protein and transgenic plants, whose phenotypes were also investigated. A OsBRI1 cytoplasmic domain (CD) recombinant protein was induced in BL21 (DE3) E.coli cells with IPTG, and purified to obtain OsBRI1 recombinant protein. Based on Western blot analysis with phospho-specific pTyr and pThr antibodies, OsBRI1 recombinant protein and OsBRI1-Flag protein were phosphorylated on Threonine residue(s), however, not on Tyrosine residue(s), both in vitro and in vivo. This is particularly intriguing as AtBRI1 protein was phosphorylated on both Ser/Thr and Tyr residues. Also, the OsBRI1 full-length gene was expressed in, and rescued, bri1-5 mutants, such as is seen in normal wild-type plants where AtBRI1-Flag rescues bri1-5 mutant plants. Root growth in seedlings decreased in Ws2, AtBRI1, and 3 independent OsBRI1 transgenic seedlings and had an almost complete lack of response to brassinolide in the bri1-5 mutant. In conclusion, OsBRI1, an orthologous gene of AtBRI1, can mediate normal BR signaling for plant growth and development in Arabidopsis thaliana.

• Journal of Life Science
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• v.9 no.5
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• pp.556-563
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• 1999

Presence of antibody to the capsid protein p24 is the main diagnostic criterion, since this reflects reliable antibody response to HIV infection. However, it takes about 6-8 weeks for antibody production after infection and people who are infected but antibodies are not produced yet are classified as seronegative. Therefore, there is a strong need for an improved diagnostic method for better health security. As a first step for developing such an improved diagnostic system, gag protein of human immunodificiency virus type 1 was expressed in E. coli DH5$\alpha$. The gag fragment of HIV-1 (including a portion of p17 and whole p24) was amplified by polymerase chain reaction (PCR) and BamH I/EcoR I sites were created during PCR. The amplified DNA fragment was cleaved with BamH I/EcoR I and was subcloned into the GEX-2T vector which had been digested with BamHI/EcoRI, resulting gene fusion with gst gene of pGEX-2T. The recombinant DNA was transferred into E. coli DH5$\alpha$. The transformed bacteria were grown at 37$^{\circ}C$ for 3h and protein expression was induced with 0.1mM IPTG at $25^{\circ}C$ for 3h. Recombinant gag protein or GST-gag fusion protein was purified with glutathione-sepharose 4B bead and migrated as a single band when analyzed by 10% polyacrylamide gel. These proteins were confirmed by immunoblotting with anti-GST goat sera or Korean AIDS patients sera. The results of this study establish the expression and single step pulification of HIV-1 gag protein which can specifically bind with Korean AIDS patients sera.

• Parasites, Hosts and Diseases
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• v.52 no.1
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• pp.93-97
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• 2014

Subolesin (4D8), the ortholog of insect akirins, is a highly conserved protective antigen and thus has the potential for development of a broad-spectrum vaccine against ticks and mosquitoes. To date, no protective antigens have been characterized nor tested as candidate vaccines against Dermacentor silvarum bites and transmission of associated pathogens. In this study, we cloned the open reading frame (ORF) of D. silvarum 4D8 cDNA (Ds4D8), which consisted of 498 bp encoding 165 amino acid residues. The results of sequence alignments and phylogenetic analysis demonstrated that D. silvarum 4D8 (Ds4D8) is highly conserved showing more than 81% identity of amino acid sequences with those of other hard ticks. Additionally, Ds4D8 containing restriction sites was ligated into the pET-32(a+) expression vector and the recombinant plasmid was transformed into Escherichia coli rosetta. The recombinant Ds4D8 (rDs4D8) was induced by isopropyl ${\beta}$-D-thiogalactopyranoside (IPTG) and purified using Ni affinity chromatography. The SDS-PAGE results showed that the molecular weight of rDs4D8 was 40 kDa, which was consistent with the expected molecular mass considering 22 kDa histidine-tagged thioredoxin (TRX) protein from the expression vector. Western blot results showed that rabbit anti-D. silvarum serum recognized the expressed rDs4D8, suggesting an immune response against rDs4D8. These results provided the basis for developing a candidate vaccine against D. silvarum ticks and transmission of associated pathogens.

• Korean Journal of Medicinal Crop Science
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• v.11 no.1
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• pp.31-39
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• 2003

The antioxidative activities and biological properties in the EtOAc extracts and purified extracts of Orostachys japonicus were measured by assay of DPPH, xanthine/xanthine oxidase and mammalian cells(2-12). Scavenging of DPPH radical and inhibition of xanthine/xanthine oxidase of EtOAc extracts were showed the highest activity in the arable land and in September. The S-4 fraction showed the highest activity among the silica-gel column chromatography fractions. LH-4 fraction showed higher activity than the other fractionsins in assay of DPPH and xanthine/xanthine oxidase. Fatty acids and phenolic compounds were identified by GC/MS and main compounds were 1,2,3-benzenetriol, alpha-androsta-7,14-diene in LH-4 fraction. The activities of POD and SOD in samples havested on different habitats were high such as arable land> intermountain> seashore. That of POD and SOD in crude extracts of late stage were higher than early stage. Isozyme bands of crude extracts samplinged in all habitats and all growing stages showed two bands and the signal intensity showed strongly according to passage of growing stage. The purified extracts of LH-4 fraction showed excellent inhibition effect in proliferation of HL-60 cells and markedly suppressed colony formation in mouse fibroblast cells. Dose response between partially purified extracts(400ppm) and negative control did not produced statistically significant reduction in colony formation.