• Journal of Microbiology and Biotechnology
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• 제16권7호
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• pp.1026-1031
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• 2006

The optimization of the production of a fusion protein containing the antigenic domain 1 (AD-1) is of a great importance, considering its use in diagnostic tests. The fusion protein is produced by the fermentation of a recombinant strain of Escherichia coli containing the plasmid Mbg58, which expresses the AD-1 (aa 484-650) of human cytomegalovirus glycoprotein B as a fusion protein together with aa 1-375 of ${\beta}-galactosidase$. An important characteristic of promoters (lac and derivatives) used in recombinant protein production in E. coli is their inducibility. Induction by IPTG is widely used for basic research; however, its use in large-scale production is undesirable because of its high cost and toxicity. In this work, studies using different inducers and carbon sources for the production of a fusion protein containing the AD-l were performed. The results showed that lactose could be used as an inducer in the fermentation process for the production of this protein, and that expression levels could exceed those achieved with IPTG. The use of lactose for protein expression in E. coli should be extremely useful for the inexpensive, large-scale production of heterologous proteins in E. coli. Addition of sucrose to the fermentation medium improved the yield of recombinant protein, whereas addition of fructose or trehalose decreased the yield.

• Journal of Microbiology
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• 제37권4호
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• pp.234-242
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• 1999

To obtain the recombinant circumsporozoite (CS) protein for the diagnosis of patients and seroepidemiology of Plasmodium vivax malaria which have been prevalent in northern part of Kyonggido, the CS protein gene was amplified by the polymerase chain reaction (PCR) from genomic DNA of the Korean vivax malaria patient. The gene consists of 1,123 nucleotides except signal peptide sequences and had an uninterrupted reading frame encoding a protein of 374 amino acids with a central region of 20 tandem repeats of the nonapeptide. The CS protein gene was expressed in Escherichia coli and purified, the molecular weight of recombinant CS protein was about 44 kDa (monomer) under denaturing purification and about 65 kDa (dimer) under native purification by SDS-PAGE. The purified recombinant CS protein which has antigenicity to malaria patients in Western blot analysis and Enzyme-linked immunosorbent assay, reacted only with the serum of P. vivax (PV210) infected malaria patients with no cross reaction to the P. falciparum malaria patient. The recombinant CS protein purified in this study will serve as a useful antigen to support the diagnosis of malaria patients and seroepidemiology.

• Journal of Microbiology and Biotechnology
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• 제25권10호
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• pp.1761-1767
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• 2015

Sevenband grouper, Epinephelus septemfasciatus, is becoming an important aquaculture species in Korea. However, viral nervous necrosis disease is a large problem causing mass mortality in sevenband grouper aquaculture. Recombinant protein vaccines are one of the best methods to reduce these economic losses. However, the cell-based expression method mainly produces inclusion bodies and requires additional procedures. In this study, we expressed a recombinant viral coat protein of sevenband grouper nervous necrosis virus (NNV) using a cell-free protein synthesis system. The purified recombinant NNV coat protein (rNNV-CP) was injected into sevenband grouper at different doses followed by a NNV challenge. Nonimmunized fish in the first trial (20 μg/fish) began to die 5 days post-challenge and reached 70% cumulative mortality. In contrast, immunized fish also starting dying 5 days postchallenge but lower cumulative mortality (10%) was observed. Cumulative morality in the second trial with different doses (20, 4, and 0.8 μg/fish) was 10%, 40%, and 50%, respectively. These results suggest that rNNV-CP can effectively immunize sevenband grouper depending on the dose administered. This study provides a new approach to develop a recombinant vaccine against NNV infection for sevenband grouper.

• 한국어병학회지
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• 제26권3호
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• pp.231-240
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• 2013

Adjuvants are immune enhancers that are often used in vaccination to augment the immune response of a vaccine, thereby enhancing the protective immunity against the targeted disease. In the present study, we used the recombinant protein, such as rRbCC1, this protein was produced from rock bream CC chemokine 1. To verify the adjuvant effects of this recombinant protein, the immune responses of rock bream to Streptococcus iniae (S. iniae) FKC vaccination, which alone or in combination with recombinant protein was analyzed and then also performed experimental challenge with live S. iniae. The result of serum agglutination titres was showed relatively low levels however, the efficacy of FKC vaccine still conferred protection against S. iniae. Moreover, the adverse effects result showed that no statistically significant difference was revealed between high concentration injected and non-injected fish groups, generally. The relative percent survival (RPS) of FKC + recombinant vaccination group was significantly higher than that of vaccinated group with FKC alone. After experimental challenge to the rock bream by injection with live bacteria (S. iniae), the FKC + rRbCC1 vaccination group was showed 87.0% RPS, however, the RPS of FKC alone vaccination was 68.2%. The results indicated that the recombinant protein as an adjuvant had a clear synergism to injection vaccine of rock bream.

• Journal of Microbiology and Biotechnology
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• 제17권11호
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• pp.1898-1903
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• 2007

We attempted to modulate the overall protein expression rate through the addition of a repressor against the araBAD promoter system of Escherichia coli, in which glucose was used as a repressor. Therefore, 0.5% L-arabinose was initially contained as an inducer in culture medium, and either 2% glucose or 2% glycerol was used as a carbon source, and it was found that the expression of recombinant interferon-${\alpha}$ could be observed at the beginning of the batch culture when glycerol was used as a carbon source. However, when glucose was used, the initiation of recombinant interferon-${\alpha}$ expression was delayed compared with that when glycerol was used. Furthermore, when the addition of 0.5% glucose was carried out once or twice after 0.5% L-arabinose induction during DO-stat fed-batch culture, the distributions of soluble and insoluble recombinant interferon-${\alpha}$ were modulated. When glucose was not added after the induction of L-arabinose, all of the expressed recombinant interferon-${\alpha}$ formed an inclusion body during the later half of culturing. However, when glucose was added after induction, the expressed recombinant interferon-${\alpha}$ did not all form an inclusion body, and about half of the total recombinant interferon-${\alpha}$ was expressed in a soluble form. It was deduced that the addition of glucose after the induction of L-arabinose might lower the cAMP level, and thus, CAP (catabolite activator protein) might not be activated. The transcription rate of recombinant interferon-${\alpha}$ in the araBAD promoter system might be delayed by the partial repression. This inhibition of the transcription rate probably resulted in more soluble interferon-${\alpha}$ expression caused by the reduction of the protein synthesis rate.

• Journal of Microbiology and Biotechnology
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• 제13권5호
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• pp.738-744
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• 2003

The gene ADH encoding NAD-dependent alcohol dehydrogenase from Bacillus stearothennophilus was cloned and overexpressed as a GST fusion protein at a high level in Escherichia coli. The expressed fusion protein was purified simply by glutathione affinity chromatography. GST fusion protein was then cleaved by thrombin, while soluble enzyme was further purified by glutathione affinity chromatography. The recombinant enzyme had the same elctrophoretic mobility as the native enzyme from Bacillus stearothennophilus. The recombinant enzyme catalyzed the oxidation of a number of alcohols and exhibited high activities towards secondary alcohols. The $K_m\;and\;V_{max}$ values of the recombinant enzyme for ethanol were 5.11 mM and 61.35 U/mg, respectively. Pyridine and imidazole notably inhibited the enzymatic activity. The activity of the recombinant enzyme optimally proceeded at pH 9.0 and $70^{\circ}C$. The midpoint of the temperature-stability curve for the recombinant enzyme was approximately $68^{\circ}C$, and the enzyme was not completely inactivated even at $85^{\circ}C$. The recombinant enzyme showed a high resistance towards denaturing agents (0.05% SDS, 0.1 M urea). Therefore, due to its stability and relatively broad substrate specificity, the recombinant enzyme could be utilized in bio-industrial processes and biosensors.

• BMB Reports
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• 제28권3호
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• pp.216-220
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• 1995

cDNA for human cholesteryl ester transfer protein (CETP), a potent atherogenic plasma protein that redistributes the neutral lipids among lipoproteins, was expressed in recombinant vaccinia virus-infected cells (CV-1). Two insertion vectors regulated by different promoters were constructed. The vectors were introduced into human thymidine kinase-negative ($TK^-$) 1438 cells infected with wild-type vaccinia virus (WR strain). Recombinant viruses were selected with 5-bromodeoxyuridine (BUdR) and X-gal and identified with DNA dot blot analysis (vSC11-CETP and vTM1-CETP). The CETP cDNA insert in the recombinant vaccinia virus genome was identified by Southern blot analysis. Transcription of CETP cDNA in CV-1 cells infected with recombinant vaccinia virus was monitored by Northern blot analysis using the CETP cDNA as a probe. Positive signals were detected at 1.8 kb in cells infected with vSC11-CETP and at 2.3 kb in cells infected with vTM1-CETP. The recombinant vaccinia virus-infected CV-1 cells were shown to produce functional CETP when the culture medium was subjected to the CETP assay.

• KSBB Journal
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• 제18권6호
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• pp.435-439
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• 2003

형질 전환된 담배 seed에서 담배 식물체를 유도하여 White 액체 배지에서 기관 배양하였다. 암조건, sucrose 2%의 조건에서 좋은 growth pattern을 나타내었고, 계대 배양은 외마디법을 이용하여 2주마다 하였다. 기관 배양에서 hGM-CSF production pattern을 보면, intracellular에서는 큰 변화 없이 약 30 ng/g의 일정한 농도를 나타내었다. Extracellular에서 hGM-CSF 농도는 배양 6일 이후부터 급속하게 증가하기 시작하여 배양 12일째에 약 0.2ng/$m\ell$의 농도를 나타낸다. 기관배양은 다른 식물세포 배양 시스템에 비해 생산되어진 단백질의 안정성이 크다는 장점에 비해 세포 내에서 배지 내로의 단백질 분비가 적다는 단점이 있다. 이를 극복하기 위해 다양한 permeabilizing agents를 투여하여 담배 세포의 permeability를 증가시키고자 하였다. 그 결과, Pluronic F-68과 PEG8000을 첨가한 경우 담배 세포에서 배지 내로의 단백질 분비가 원활해졌음을 확인할 수 있었다.

• International Journal of Industrial Entomology and Biomaterials
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• 제32권2호
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• pp.49-53
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• 2016

Bone morphogenetic proteins (BMPs) are essential growth factors for bone formation, skeletal development and bone regeneration. The BMP-2/7 heterodimer is known to have remarkable effects on osteogenic induction that are even stronger than the BMP-2 or BMP-7 homodimers. We designed a recombinant human BMP-2/7 (rhBMP-2/7) heterodimer protein with four glycine residues between BMP-2 and BMP-7 protein to facilitate free bond rotation of domains. The Baculovirus Expression Vector System (BEVS) is routinely used to produce recombinant proteins in the milligram scale. In this study, the BEVS was used to express the rhBMP-2/7 protein whrer the recombinant baculovirus was recovered in the host Sf9 cells. To confirm the biological activity of rhBMP-2/7 protein secreted from the BEVS as an osteogenic differentiation and induction factor, we measured the BMP-induced ALP activity. rhBMP-2/7 could be used as an alternative to BMPs to overcome limitations like short half-life and requirement for high concentrations. Furthermore, rhBMP-2/7 may be an efficient tool for various application studies such as bone regeneration and skeletal development.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권4호
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• pp.275-287
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• 2000

Fermentation strategies for recombinant protein production in Pichia pastoris have been investigated and are reviewed here. Characteristics of the expression system, such as phenotypes and carbon utilization, are summarized. Recently reported results such as growth model establishment, app58lication of a methanol sensor, optimization of substrate feeding strategy, DOstat controller design, mixed feed technology, and perfusion and continuous culture are discussed in detail.