• 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.

• Proceedings of the Polymer Society of Korea Conference
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• 2006.10a
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• pp.256-256
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• 2006

The design and synthesis of novel biomolecular materials, based on mimicking the properties of molecules found in nature, is providing materials with unusual properties. Resilin serves as an energy storage material in insects and facilitates flight, jumping (in fleas, froghoppers etc) and sound production (cicadas, etc). Resilin is initially produced as a soluble protein and in its mature form is crosslinked through formation of dityrosine units into a very large insoluble polymer. In the present study, we have synthesized a recombinant form of resilin that can be photochemically cross-linked into a resilient, rubber-like biomaterial that may be suitable for spinal disc implants. This material is almost perfectly elastic and its fatigue lifetime in insects must be >500 million cycles.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.789-793
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• 1999

The effect of acetic acid formation deficiency on recombinant E. coli fermentation was investigated using a mutant strain deficient in acetic acid formation. A mutant strain which does not grow under anaerobic conditions was isolated. The acetic acid production in this strain was negligible in aerobic batch fermentation. The cloned-gene expression in the mutant strain was higher than the wild-type strain. Fed-batch fermentations with controlled specific growth rates were carried out in order to compare the cloned-gene expression between the wild-type and the mutant strains. The expression decreased along with the specific growth rate in both strains. The cloned-gene expression in the mutant strain was 60% higher than in the wild-type strain at the same specific growth rate.

• Proceedings of the PSK Conference
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• 2003.10b
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• pp.170.3-171
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• 2003

An efficient Erythropoietin (EPO)-expression system in mammalian cells is required for massive production for therapeutic use. Ammonium ion is a major problem in the production of valuable recombinant proteins in cultured animal cells. Therefore, it is of importance to devise a system by which a high productivity of human therapeutic recombinant protein can be maintained or enhanced under low ammonium concentration. To reduce the ammonium ion accumulated in EPO producing Chinese Hamster Ovary (CHO) ceels, IBE, we introduced the first two genes of the urea cycle, carbamoyl phosphate synthetase (CPSI) and arnithine transcarbamoylase (OTC), into IBE using a stable transfection method. (omitted)

• Journal of Microbiology and Biotechnology
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• v.23 no.8
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• pp.1047-1054
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• 2013

Witches' broom of lime is a disease caused by Candidatus Phytoplasma aurantifolia, which represents the most significant global threat to the production of lime trees (Citrus aurantifolia). Conventional disease management strategies have shown little success, and new approaches based on genetic engineering need to be considered. The expression of recombinant antibodies and fragments thereof in plant cells is a powerful approach that can be used to suppress plant pathogens. We have developed a single-chain variable fragment antibody (scFvIMP6) against the immunodominant membrane protein (IMP) of witches' broom phytoplasma and expressed it in different plant cell compartments. We isolated scFvIMP6 from a naïve scFv phage display library and expressed it in bacteria to demonstrate its binding activity against both recombinant IMP and intact phytoplasma cells. The expression of scFvIMP6 in plants was evaluated by transferring the scFvIMP6 cDNA to plant expression vectors featuring constitutive or phloem specific promoters in cassettes with or without secretion signals, therefore causing the protein to accumulate either in the cytosol or apoplast. All constructs were transiently expressed in Nicotiana benthamiana by agroinfiltration, and antibodies of the anticipated size were detected by immunoblotting. Plant-derived scFvIMP6 was purified by affinity chromatography, and specific binding to recombinant IMP was demonstrated by enzyme-linked immunosorbent assay. Our results indicate that scFvIMP6 binds with high activity and can be used for the detection of Ca. Phytoplasma aurantifolia and is also a suitable candidate for stable expression in lime trees to suppress witches' broom of lime.

• International Journal of Industrial Entomology and Biomaterials
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• v.28 no.1
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• pp.10-18
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• 2014

Baculoviruses base vectors come to be regarded as methods for in vivo gene delivery and transient expression to the silkworm. In the case of silkworm, B. mori, two types of baculoviruses, AcNPV (Autographa californica nuclear polyhedrosis virus) and BmNPV (Bombyx mori nuclear polyhedrosis virus), are potentially applicable as vectors. Recently, AcNPV showed promising results with some silkworm strains despite different host-specificities. We searched for a highly-permissive silkworm strain in the B. mori stocks of Kyungpook National University that could produce high levels of recombinant protein. Seventy strains were screened using the recombinant AcNPV/BmA3-Luc virus. Based on the measured luciferase activity, the strains could be divided into three groups, high-, middle-, and low-permissive strains, according to their relative recombinant protein expression levels. At 48 hours post-injection, the luciferase activity in the high-permissive strains was 500-fold greater than that of the low-permissive strains. At 72 hours post-injection, a significant elevation in luciferase activity was observed in the hemocytes of all strains. Then, based on the above results, the High Permissive Strain (HPS) S10 and the Low Permissive Strain (LPS) S39 were pick up and was carried out Dot blotting, RT-PCR and Real time PCR.

• Journal of Periodontal and Implant Science
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• v.38 no.sup2
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• pp.335-342
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• 2008

Purpose: Cytolethal distending toxin (CDT) considered as a key factor of localized aggressive periodontitis, endocarditis, meningitis, and osteomyelitis is composed of five open reading frames (ORFs). Among of them, the individual role of CdtA and CdtC is not clear; several reports presents that CDT is an AB2 toxin and they enters the host cell via clathrin-coated pits or through the interaction with GM3 ganglioside. So, CdtA, CdtC, or both seem to be required for the delivery of the CdtB protein into the host cell. Moreover, recombinant CDT was suggested as good vaccine material and antibody against CDT can be used for neutralization or for a detection kit. Materials and Methods: We constructed the pET28a-cdtC plasmid from Aggregatibacter actinomycetemcomitans Y4 by genomic DNA PCR and expressed in BL21 (DE3) Escherichia coli system. We obtained the antibody against the recombinant CdtC in mice system. Using the anti-CdtC antibody, we test the native CdtC detection by ELISA and Western Blotting and confirm the expression time of native CdtC protein during the growth phase of A. actinomycetemcomitans. Results: In this study we reconstructed CdtC subunit of A. actinomycetemcomitans Y4 and generated the anti CdtC antibody against recombinant CdtC subunit expressed in E. coli system. Our anti CdtC antibody can be interacting with recombinant CdtC and native CDT in ELISA and Western system. Also, CDT holotoxin existed at 24h but not at 48h meaning that CDT holotoxin was assembled at specific time during the bacterial growth. Conclusion: In conclusion, we thought that our anti CdtC antibody could be used mucosal adjuvant or detection kit development, because it could interact with native CDT holotoxin.

• Journal of Microbiology and Biotechnology
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• v.24 no.12
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• pp.1685-1689
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• 2014

Baeyer-Villiger (BV) oxidation of cyclohexanone to ${\varepsilon}$-caprolactone in a microbial system expressing cyclohexanone monooxygenase (CHMO) can be influenced by not only the efficient regeneration of NADPH but also a sufficient supply of oxygen. In this study, the bacterial hemoglobin gene from Vitreoscilla stercoraria (vhb) was introduced into the recombinant Escherichia coli expressing CHMO to investigate the effects of an oxygen-carrying protein on microbial BV oxidation of cyclohexanone. Coexpression of Vhb allowed the recombinant E. coli strain to produce a maximum ${\varepsilon}$-caprolactone concentration of 15.7 g/l in a fed-batch BV oxidation of cyclohexanone, which corresponded to a 43% improvement compared with the control strain expressing CHMO only under the same conditions.

• Journal of Microbiology and Biotechnology
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• v.21 no.10
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• pp.1053-1056
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• 2011

Optimal conditions for a high cell density fermentation were investigated in a recombinant Escherichia coli producing salmosin, a platelet aggregation inhibitor. The optimized carbon and nitrogen sources were glycerol 10 g/l, yeast extract 30 g/l, and bacto-tryptone 10 g/l, yielding the dry cell weight (DCW) of 10.61 g/l in a 500 ml flask culture. The late-stage induction with 1% L-arabinose in a 5 l jar fermentor showed the highest DCW of 65.70 g/l after 27 h of the fed-batch fermentation. Around 2,200 mg/l of the protein was expressed as an inclusion body that was then refolded to obtain the active salmosin of 96 mg/l. We also confirmed the inhibitory activity against platelet aggregation of the active salmosin from the high cell density fermentation.

• Korean Journal of Agricultural Science
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• v.41 no.3
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• pp.245-249
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• 2014

Among dozens of DNA polymerases cloned from thermophilic bacteria, Taq DNA polymerase from Thermus aquaticus has been most frequently used in polymerase chain reaction (PCR) that is being applied to gene cloning, DNA sequencing, gene expression analysis, and detection of infectious and genetic diseases. Since native Taq DNA polymerase is expressed at low level in T. aquaticus, recombinant Escherichia coli system was used to produce Taq DNA polymerase in a large amount. Taq DNA polymerase was expressed as a soluble form under the control of tac promoter in E. coli, and purified by heat treatment and ion exchange chromatographies. The purified Taq DNA polymerase was nearly homogeneous and exhibited a similar DNA amplification activity with a commercial Taq DNA polymerase.