Hong Weon Lee

Korea Research Institute of Bioscience and Biotechnology KRIBB, Anzan, Gyeonggi Province, South Korea

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Publications (8)17.89 Total impact

  • Shin-Young Kang · Yeon-Gu Kim · Hong Weon Lee · Eun Gyo Lee ·
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    ABSTRACT: Gene amplification using dihydrofolate reductase gene (dhfr) and methotrexate (MTX) is widely used for recombinant protein production in mammalian cells and is typically conducted in DHFR-deficient Chinese hamster ovary (CHO) cell lines. Generation of DHFR-deficient cells can be achieved by an expression vector incorporating short hairpin RNA (shRNA) that targets the 3'-untranslated region (UTR) of endogenous dhfr. Thus, shRNAs were designed to target the 3'-UTR of endogenous dhfr, and shRNA-2 efficiently down-regulated dhfr expression in CHO-K1 cells. A single gene copy of shRNA-2 also decreased the translational level of DHFR by 80 % in Flp-In CHO cells. shRNA-2 was then incorporated into a plasmid vector expressing human erythropoietin (EPO) and an exogenous DHFR to develop EPO-producing cells in the Flp-In system. The specific EPO productivity (q EPO) was enhanced by stepwise increments of MTX concentration, and differences in the amplification rate were observed in Flp-In CHO cells that expressed shRNA-2. In addition, the q EPO increased by more than 2.5-fold in the presence of 500 nM MTX. The mRNA expression level and gene copy numbers of dhfr were correlated with increased productivity in the cells, which is influenced by inhibition of endogenous dhfr. This study reveals that an expression vector including shRNA that targets the 3'-UTR of endogenous dhfr can enhance the transgene amplification rate and productivity by generating DHFR-deficient cells. This approach may be applied for amplifying the foreign gene in wild-type cell lines as a versatile single-plasmid vector.
    Applied Microbiology and Biotechnology 08/2015; DOI:10.1007/s00253-015-6856-y · 3.34 Impact Factor
  • Sasikumar Rajoo · Jung Oh Ahn · Hong Weon Lee · Joon Ki Jung ·
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    ABSTRACT: For the isolation of a ε-caprolactam-degrading microbe from wastewaters of a factory producing caprolactam, we applied a chemostat-enrichment technique with a selective medium containing caprolactam as sole source of carbon and nitrogen. This allowed for the isolation of a novel caprolactam-degrading microbe, identified as Acinetobacter calcoaceticus. The strain had a critical tolerance of 19 g caprolactam l(-1) in minimal medium, which is higher than any previously reported caprolactam-degrading microbe. A. calcoaceticus also decreased the caprolactam content in medium by 65 % within 72 h despite the high caprolactam content (10 g l(-1)). This study highlights the potential use of A. calcoaceticus strain for the bioremediation of recalcitrant synthetic polymers, such as caprolactam.
    Biotechnology Letters 08/2013; 35(12). DOI:10.1007/s10529-013-1307-2 · 1.59 Impact Factor
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    ABSTRACT: Overexpression of Bcl-2, a typical anti-apoptotic protein, is one of the most effective means to maintain mitochondria integrity in recombinant CHO (rCHO) cell culture treated with sodium butyrate (NaBu). NaBu is known as a typical specific productivity-enhancing factor and also a well-known apoptosis inducer. Bcl-2 is distributed to and functions in multiple intracellular organelles such as the nucleus, mitochondria, and endoplasmic reticulum (ER). To evaluate the effect of organelle-specific overexpression of Bcl-2 on NaBu-induced apoptosis in rCHO cells, Bcl-2 expression was restricted to the mitochondria or to the ER either by employing a mitochondrial insertion sequence of ActA or by insertion of an ER-specific sequence of cytochrome b5 to their respective sequences. The rCHO cell lines overexpressing wild-type Bcl-2 (WT-Bcl-2), mitochondrial Bcl-2 (MT-Bcl-2), and ER-targeted Bcl-2 (ER-Bcl-2) were established. Overexpression of WT-Bcl-2, MT-Bcl-2, and ER-Bcl-2 could increase cell viability and decrease LDH release under NaBu-treated conditions. Additionally, overexpression of WT-Bcl-2, MT-Bcl-2, and ER-Bcl-2 could suppress NaBu-induced apoptosis, as demonstrated by a DNA fragmentation assay. A mitochondrial membrane potential assay revealed that ER-Bcl-2 overexpression can maintain the mitochondrial membrane integrity without being affected by MT-Bcl-2 overexpression, indicating that the role of ER should be considered in alleviating NaBu-induced apoptosis by a genetic modulation strategy. Taken together, it was found that restricted Bcl-2 overexpression at the ER can inhibit the NaBu-induced apoptosis by maintaining mitochondria integrity in rCHO cells.
    PROCESS BIOCHEMISTRY 12/2012; 47(12):2518-2522. DOI:10.1016/j.procbio.2012.06.021 · 2.52 Impact Factor
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    ABSTRACT: The establishment of high producer is an important issue in Chinese hamster ovary (CHO) cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS)-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production. An internal ribosome entry site (IRES) was introduced for using two green fluorescence protein (GFP) fragments as a reporter to both antibody chains, the heavy chain and the light chain. The cells co-transfected with two GFP fragments showed the emission of green fluorescence by the reconstitution of split GFP. The FACS-sorted pool with GFP expression had a higher specific antibody productivity (q(Ab)) than that of the unsorted pool. The q(Ab) was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and q(Ab) in individual selected clones. This study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of q(Ab) with reconstitution of GFP. Taken together, we developed an efficient FACS-based screening method for high antibody-producing CHO cells with the benefits of the split GFP system.
    BMC Biotechnology 05/2012; 12(1):24. DOI:10.1186/1472-6750-12-24 · 2.03 Impact Factor
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    ABSTRACT: Dimethyl sulfoxide (DMSO) can increase the specific productivity (q) of foreign proteins in mammalian cells, while it can also induce cell death, particularly apoptosis. Bcl-xL is a typical anti-apoptotic protein that inhibits the apoptosis in recombinant Chinese hamster ovary (rCHO) cell culture. To evaluate the potential role of Bcl-xL overexpression on DMSO-mediated erythropoietin (EPO) production, we used EPO-producing rCHO cells with regulated Bcl-xL overexpression (EPO-off-Bcl-xL) by doxycycline. Although DMSO addition enhanced specific EPO productivity (qEPO), it also induced cell death in EPO-off-Bcl-xL cells. Bcl-xL overexpression reduced the DMSO-induced cell death followed by release of various enzymes from plasma membrane-damaged cells as evidenced from LDH assay, resulting in delayed loss of EPO. However, it did not significantly improve the maximum EPO production. In addition, Bcl-xL overexpression suppressed DMSO-induced apoptosis, characterized by DNA fragmentation and Annexin V staining. Taken together, Bcl-xL overexpression could inhibit DMSO-induced apoptosis, thereby delaying the loss of EPO.
    PROCESS BIOCHEMISTRY 11/2011; 46(11):2201-2204. DOI:10.1016/j.procbio.2011.07.017 · 2.52 Impact Factor
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    ABSTRACT: Constitutively active Ras (CA-Ras) is known to enhance cell growth through the induction of various signaling cascades including the phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/ERK signaling pathways, although the cellular response is highly dependent on the cell type. To evaluate the effect of CA-Ras overexpression on cell growth in recombinant Chinese hamster ovary (rCHO) cells, an erythropoietin (EPO)-producing rCHO cell line with regulated CA-Ras overexpression (EPO-off-CA-Ras) was established using the Tet-off system. The CA-Ras expression level in EPO-off-CA-Ras cells was tightly regulated by doxycycline addition. Although CA-Ras overexpression slightly increased the viable cell concentration during the late exponential phase, it did not increase the maximum viable cell concentration or specific growth rate to a significant degree. Unexpectedly, CA-Ras overexpression in rCHO cells led only to the enhancement in the activation of the MAPK/ERK signaling pathway and not the PI3K/Akt signaling pathway. Taken together, CA-Ras overexpression in rCHO cells did not significantly affect cell growth; it also had no critical impact on viable cell concentration or EPO production, possibly due to a failure to activate the PI3K/Akt signaling pathway.
    Biotechnology Progress 03/2011; 27(2):577-80. DOI:10.1002/btpr.567 · 2.15 Impact Factor
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    ABSTRACT: We describe a method of amplifying the biosensing signal in surface plasmon resonance (SPR)-based immunoassays using an antibody-carbon nanotube (CNT) conjugate. As a model system, human erythropoietin (EPO) and human granulocyte macrophage colony-stimulating factor (GM-CSF) were detected by sandwich-type immunoassays using an SPR biosensor. For the amplification of the SPR signal, the CNT was conjugated with a polyclonal antibody, and then the conjugates were reacted with antibodies coupled with the target proteins. This amplification strategy increases the dynamic range of the immunoassays and enhances the detection sensitivity. The SPR immunoassays, combined with the CNT-assisted signal amplification method, provided a wide dynamic range over four orders of magnitude for both EPO and GM-CSF (0.1-1,000 ng/ml). The CNT amplification method is expected to realize the detection of picogram levels and a wide dynamic detection range of multiple proteins, enabling it to offer a robust analysis tool for the development of biopharmaceutical production.
    Analytical Biochemistry 01/2011; 408(2):206-11. DOI:10.1016/j.ab.2010.09.026 · 2.22 Impact Factor
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    ABSTRACT: Effects of various industrially important carbon sources (glucose, sucrose, xylose, gluconate, and glycerol) on shikimic acid (SA) biosynthesis in Escherichia coli were investigated to gain new insight into the metabolic capability for overproducing SA. At the outset, constraints-based flux analysis using the genome-scale in silico model of E. coli was conducted to quantify the theoretical maximum SA yield. The corresponding flux distributions fueled by different carbon sources under investigation were compared with respect to theoretical yield and energy utilization, thereby identifying the indispensable pathways for achieving optimal SA production on each carbon source. Subsequently, a shikimate-kinase-deficient E. coli mutant was developed by blocking the aromatic amino acid pathway, and the production of SA on various carbon sources was experimentally examined during 5 l batch culture. As a result, the highest production rate, 1.92 mmol SA/h, was obtained when glucose was utilized as a carbon source, whereas the efficient SA production from glycerol was obtained with the highest yield, 0.21 mol SA formed per mol carbon atom of carbon source consumed. The current strain can be further improved to satisfy the theoretically achievable SA production that was predicted by in silico analysis.
    Journal of Microbiology and Biotechnology 12/2008; 18(11):1773-84. · 1.53 Impact Factor