• Journal of Microbiology and Biotechnology
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• 제24권2호
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• pp.152-159
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• 2014

The regulation of protein tyrosine phosphorylation is mediated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) and is essential for cellular homeostasis. Co-expression of PTKs with PTPs in Pichia pastoris was used to facilitate the expression of active PTKs by neutralizing their apparent toxicity to cells. In this study, the gene encoding phosphatase PTP1B with or without a blue fluorescent protein or peroxisomal targeting signal 1 was cloned into the expression vector pAG32 to produce four vectors. These vectors were subsequently transformed into P. pastoris GS115. The tyrosine kinases EGFR-2 and $PDGFR{\beta}$ were expressed from vector pPIC3.5K and were fused with a His-tag and green fluorescent protein at the N-terminus. The two plasmids were transformed into P. pastoris with or without PTP1B, resulting in 10 strains. The EGFR-2 and $PDGFR{\beta}$ fusion proteins were purified by $Ni^{2+}$ affinity chromatography. In the recombinant P. pastoris, the PTKs co-expressed with PTP1B exhibited higher kinase catalytic activity than did those expressing the PTKs alone. The highest activities were achieved by targeting the PTKs and PTP1B into peroxisomes. Therefore, the EGFR-2 and $PDGFR{\beta}$ fusion proteins expressed in P. pastoris may be attractive drug screening targets for anticancer therapeutics.

• Journal of Microbiology and Biotechnology
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• 제24권4호
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• pp.431-439
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• 2014

We report the construction of two Bacillus subtilis expression vectors, pBNS1/pBNS2. Both vectors are based on the strong promoter P43 and the ampicillin resistance gene expression cassette. Additionally, a fragment with the Shine-Dalgarno sequence and a multiple cloning site (BamHI, SalI, SacI, XhoI, PstI, SphI) were inserted. The coding region for the amyQ (encoding an amylase) signal peptide was fused to the promoter P43 of pBNS1 to construct the secreted expression vector pBNS2. The applicability of vectors was tested by first generating the expression vectors pBNS1-GFP/pBNS2-GFP and then detecting for green fluorescent protein gene expression. Next, the mannanase gene from B. pumilus Nsic-2 was fused to vector pBNS2 and we measured the mannanase activity in the supernatant. The mannanase total enzyme activity was 8.65 U/ml, which was 6 times higher than that of the parent strain. Our work provides a feasible way to achieve an effective transformation system for gene expression in B. subtilis and is the first report to achieve B. pumilus mannanase secretory expression in B. subtilis.

• Journal of Ginseng Research
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• 제43권1호
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• pp.86-94
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• 2019

Background: Ginseng is believed to have antitumor activity. Autophagy is largely a prosurvival cellular process that is activated in response to cellular stressors, including cytotoxic chemotherapy; therefore, agents that inhibit autophagy can be used as chemosensitizers in cancer treatment. We examined the ability of Korean Red Ginseng extract (RGE) to prevent autophagic flux and to make hepatocellular carcinoma (HCC) cells become more sensitive to doxorubicin. Methods: The cytotoxic effects of total RGE or its saponin fraction (RGS) on HCC cells were examined by the lactate dehydrogenase assay in a dose- or time-dependent manner. The effect of RGE or RGS on autophagy was measured by analyzing microtubule-associated protein 1A/1B-light chain (LC)3-II expression and LC3 puncta formation in HCC cells. Late-stage autophagy suppression was tested using tandem-labeled green fluorescent protein (GFP)-monomeric red fluorescent protein (mRFP)-LC3. Results: RGE markedly increased the amount of LC3-II, but green and red puncta in tandem-labeled GFP-mRFP-LC3 remained colocalized over time, indicating that RGE inhibited autophagy at a late stage. Suppression of autophagy through knockdown of key ATG genes increased doxorubicin-induced cell death, suggesting that autophagy induced by doxorubicin has a protective function in HCC. Finally, RGE and RGS markedly sensitized HCC cells, (but not normal liver cells), to doxorubicin-induced cell death. Conclusion: Our data suggest that inhibition of late-stage autophagic flux by RGE is important for its potentiation of doxorubicin-induced cancer cell death. Therapy combining RGE with doxorubicin could serve as an effective strategy in the treatment of HCC.

• 한국양식학회:학술대회논문집
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• 한국양식학회 2006년도 수산관련학회 공동학술대회 발표요지집
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• pp.476-478
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• 2006

To analyze subcellular localization of betanodavirus protein B2, a plasmid expressing Betanodavirus protein B2 fused to enhanced green fluorescent protein (EGFP-Nl) was constructed. The transient expression of full-length B2 fused to EGFP in GF cells confirmed the equal distribution of protein B2 between cytoplasm and nucleus. However, transfection of N-terminal half of the B2 revealed that this truncated form predominantly localized to the cytoplasm. By using several deletion mutants and point mutants, we determined the regions and/or motif responsible for the subcellular localization of betanodavirus.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.630-633
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• 2003

Transfer of an electron from one site to another in a molecular or between molecules and/or electrodes is one of the most fundamental and ubiquitous processes in chemistry, biology and physics. In this study fusion proteins composed by green fluorescent protein(GFP) and cytochrome b562 were used in fabricating molecular array as an electron sensitizer and electron acceptor, Protein formation onto the substrate was performed by the self-assembly technique. The fusion protein film were analyzed using scanning probe microscope(SPM), Surface Plasmon Resornance(SPR) and hybrid STM/I-V. The results suggest that the proposed molecular photodiode can be used as a basic unit of the memory device.

• BMB Reports
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• 제43권12호
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• pp.795-800
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• 2010

Huntingtin-interacting protein 1-related (HIP1r) is known to function in clathrin-mediated endocytosis and regulation of the actin cytoskeleton, which occurs continuously in non-dividing cells. This study reports a new function for HIP1r in mitosis. Green fluorescent protein-fused HIP1r localizes to the mitotic spindles. Depletion of HIP1r by RNA interference induces misalignment of chromosomes and prolonged mitosis, which is associated with decreased proliferation of HIP1r-deficeint cells. Chromosome misalignment leads to missegregation and ultimately production of multinucleated cells. Depletion of HIP1r causes persistent activation of the spindle checkpoint in misaligned chromosomes. These findings suggest that HIP1r plays an important role in regulating the attachment of spindle microtubules to chromosomes during mitosis, an event that is required for accurate congression and segregation of chromosomes. This finding may provide new insights that improve the understanding of various human diseases involving HIP1r as well as its fusion genes.

• International Journal of Oral Biology
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• 제32권1호
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• pp.7-11
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• 2007

It is difficult to introduce DNA in non-invasive manner into oral cancer cells as well as primary cells for gene manipulation and expression in vivo. So far, several methods for a gene delivery have been performed to solve this problem. Magnetofection is one of the recent methods for gene transfer, and nanoparticles are applied under a magnetic field for DNA delivery. We investigated whether the magnetofection increases the efficiency of a gene delivery into several oral cell lines. By using a plasmid coding the green fluorescent protein (GFP), the efficiency of gene transfer by magnetofection was compared with those by using the calcium phosphate and the commercial transfection agent. Indeed, the magnetofection increased the green fluorescent signal in cells, suggested that this method apparently enhance the efficiency of gene delivery without any defects in various oral cancer cell lines. Finally, we have shown that magnetofection can be a useful technique for gene delivery to difficult-to-transfect cells to perform a functional study of genes in vivo.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.524-526
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• 2003

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2005년도 제45회 학술심포지움 및 추계학술대회
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• pp.67-67
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• 2005

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.239-239
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• 2004

본 연구에서는 효과적인 유전자를 발현시킬 수 있는 retrovirus vector system을 구축하기 위하여 네 가지 promoter를 비교하였다. 일반적으로 사용되고 있는 RSV (Rous sarcoma virus), UbC (Ubiquitin), β-actin, CMV(cytomegalovirus) promoter 하에 GFP (green fluorescent protein)를 표지유전자로 사용하였다. 또한 WPRE (Woodchuck hepatitis virus Posttranscriptional Regulatory Element) 서열을 도입하여 각각의 promoter 하에서 GFP 유전자의 발현 증가 여부를 검정하였다. (중략)