• Proceedings of the PSK Conference
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• 2002.10a
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• pp.408.2-409
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• 2002

The aim of this study is to prepare biodegradable microspheres without use of any kind of surfactants or emulsifiers for a novel sustained delivery carriers of protein drugs. Poly(e-caprolactone)/poly(ethylene glycol)/poly(e-caprolactone) (CEC) triblock copolymer was synthesized by ring-opening of e-caprolactone with dihydroxy poly(ethylene glycol) and was used to make surfactant-free microspheres. (omitted)

• Molecules and Cells
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• v.42 no.2
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• pp.123-134
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• 2019

Lysophosphatidic acid (LPA) is an endogenous lysophospholipid with signaling properties outside of the cell and it signals through specific G protein-coupled receptors, known as $LPA_{1-6}$. For one of its receptors, $LPA_1$ (gene name Lpar1), details on the cis-acting elements for transcriptional control have not been defined. Using 5'RACE analysis, we report the identification of an alternative transcription start site of mouse Lpar1 and characterize approximately 3,500 bp of non-coding flanking sequence 5' of mouse Lpar1 gene for promoter activity. Transient transfection of cells derived from mouse neocortical neuroblasts with constructs from the 5' regions of mouse Lpar1 gene revealed the region between -248 to +225 serving as the basal promoter for Lpar1. This region also lacks a TATA box. For the region between -761 to -248, a negative regulatory element affected the basal expression of Lpar1. This region has three E-box sequences and mutagenesis of these E-boxes, followed by transient expression, demonstrated that two of the E-boxes act as negative modulators of Lpar1. One of these E-box sequences bound the HeLa E-box binding protein (HEB), and modulation of HEB levels in the transfected cells regulated the transcription of the reporter gene. Based on our data, we propose that HEB may be required for a proper regulation of Lpar1 expression in the embryonic neocortical neuroblast cells and to affect its function in both normal brain development and disease settings.

• Journal of Microbiology and Biotechnology
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• v.29 no.2
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• pp.274-282
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• 2019

Mercury-resistant ($Hg^R$) bacteria were isolated from heavy metal polluted wastewater and soil collected near to tanneries of district Kasur, Pakistan. Bacterial isolates AZ-1, AZ-2 and AZ-3 showed resistance up to $40{\mu}g/ml$ against mercuric chloride ($HgCl_2$). 16S rDNA ribotyping and phylogenetic analysis were performed for the characterization of selected isolates as Bacillus sp. AZ-1 (KT270477), Bacillus cereus AZ-2 (KT270478) and Bacillus cereus AZ-3 (KT270479). Phylogenetic relationship on the basis of merA nucleotide sequence confirmed 51-100% homology with the corresponding region of the merA gene of already reported mercury-resistant Gram-positive bacteria. The merE gene involved in the transportation of elemental mercury ($Hg^0$) via cell membrane was cloned for the first time into pHLV vector and transformed in overexpressed C43(DE3) E. coli cells. The recombinant plasmid (pHLMerE) was expressed and the native MerE protein was obtained after thrombin cleavage by size exclusion chromatography (SEC). The purification of fusion/recombinant and native protein MerE by Ni-NTA column, dialysis and fast protein liquid chromatography (FPLC/SEC) involved unfolding/refolding techniques. A small-scale reservoir of wastewater containing $30{\mu}g/ml$ of $HgCl_2$ was designed to check the detoxification ability of selected strains. It resulted in 83% detoxification of mercury by B. cereus AZ-2 and B. cereus AZ-3, and 76% detoxification by Bacillus sp. AZ-1 respectively (p < 0.05).

• Journal of Microbiology and Biotechnology
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• v.19 no.7
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• pp.727-733
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• 2009

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-n-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite,N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other Histagged proteins expressed in E. coli.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.421-424
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• 1992

Escherichia coli JM83 harboring penicilin G acylase gene of Bacillus megaterium ATCC14945 produced a protein in large amount (>20% of the total protein). The protein was identified as GroEL, one of the E. coli heat shock protein, by N-terminal amino acid sequence analysis. It was found that GroEL was induced by the expressed foreign penicilin G acylase at both 27 and $37^{\circ}C$.

• Journal of Photoscience
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• v.9 no.2
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• pp.382-384
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• 2002

When illuminated with strong visible light, the reaction center Dl protein of photo system II is photodamage and degraded. Reactive oxygen species and endogenous cationic radicals generated by photochemical reactions are the cause of the damage to the Dl protein. Recently we found that the photodamaged Dl protein cross-links with the surrounding polypeptides such as D2 and CP43 in photosystem II. As the cross-linking reaction is dependent on the presence of oxygen, reactive oxygen species are suggested to be involved. Among the reactive oxygen species examined, ？ OH was most effective in the formation of the cross-linked products. These results indicate that the cross-linking is mostly due to ？ OH generated at photosystem II. The cross-linking site of the Dl protein is not known. As several tyrosine residues exist at the D­E loop of the Dl protein, there is a possibility that di-Tyr is formed between the D­E loop of the Dl protein and surrounding polypeptides during the strong illumination. Therefore, we examined the formation of di-Tyr using the monoclonal antibody against di-Tyr under excess illumination of the photosystem II membranes. The results obtained here suggest that no di-Tyr is formed during the excess illumination of photosystem II.

• Korean Journal of Materials Research
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• v.28 no.3
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• pp.182-188
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• 2018

Nitrogen (N)-doped protein-based carbon as platinum (Pt) catalyst supports from tofu for oxygen reduction reactions are synthesized using a carbonization and reduction method. We successfully prepare 5 wt% Pt@N-doped protein-based carbon, 10 wt% Pt@N-doped protein-based carbon, and 20 wt% Pt@N-doped protein-based carbon. The morphology and structure of the samples are characterized by field emission scanning electron microscopy and transmission electron micro scopy, and crystllinities and chemical bonding are identified using X-ray diffraction and X-ray photoelectron spectroscopy. The oxygen reduction reaction are measured using a linear sweep voltammogram and cyclic voltammetry. Among the samples, 10 wt% Pt@N-doped protein-based carbon exhibits exellent electrochemical performance with a high onset potential of 0.62 V, a high $E_{1/2}$ of 0.55 V, and a low ${\Delta}E_{1/2}=0.32mV$. Specifically, as compared to the commercial Pt/C, the 10 wt% Pt@N-doped protein-based carbon had a similar oxygen reduction reaction perfomance and improved electrochemical stability.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.93-93
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• 1997

Analysis of protein is often frustrated by the inability to isolate large amounts of purified protein from a native source. To overcome this problem, fusion protein expression systems such as pGEX system have been widely used. Using pGEX system, the desired protein could be easily obtained in a large amount in E. coli, and then the fusion protein could be used for the study of the function of the given protein. To analyze and purify the GST fusion protein, anti-GST antibody could be used as one of the system of choice. However, the production and characterization of monoclonal anti-GST antibody has not been studied extensively yet. To produce monoclonal anti-GST antibody, GST was purified from E. coli transformed with pGEX-cs, one of the pGEX system and was used as an antigen. The monoclonal antibody was produced by fusion of the immunized spleen cells with SP2-0 myeloma cells. The antibody was characterized by ELISA, western blotting, etc. The monoclonal antibody produced in this study (mAb-GSTA) showed strong and specific immunoreactivity against not only GST but also GST-fusion proteins. Also, mAb-GSTA was successfully used for the immunoaffinity purification of the GST ${\beta}$-Rc.-third intracellular-loop fusion protein. The results of the present study suggest that mAb-GSTA may be used for the identification and purification of GST fusion proteins.

• Journal of Microbiology and Biotechnology
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• v.9 no.2
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• pp.206-212
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• 1999

The gene orf7(oxi III) was expressed using an E. coli system in anticipation that it would encode dTDP-glucose 4,6-dehydratase which is involved in the biosynthesis of the olivose moiety of chlorothricin produced from Streptomyces antibioticus Tu99. The solubility of the expressed protein increased up to 20% under optimal induction conditions. The expressed protein was purified from the E. coli BL 21(DE3) cell lysate by a 28.5-fold purification in two chromatography steps with a 38% recovery to near homogeneity. The molecular weight and N-terminal amino acid sequence of the purified protein correlated with the predicted mass and sequence deduced from the orf7 gene. The purified protein was a homodimer with a subunit relative molecular weight of 38,000 Dalton. The expressed protein was found to exhibit dTDP-glucose 4,6-dehydratase activity and be highly specific for dTDP-glucose as a substrate. The values of K'm and V'max for dTDP-glucose were 28 $\mu$M and 295 nmol $min^{-1} (mg protein)^{-1}$, respectively. dTTP and dTDP were strong inhibitors of this enzyme.$NAD^+$, the coenzyme for dTDP-glucose 4,6-dehydratase, was tightly bound to the expressed protein.

• Journal of Microbiology and Biotechnology
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• v.24 no.4
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• pp.568-576
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• 2014

TIM-1 (also known as KIM-1 and HAVcr-1) is a type I transmembrane glycoprotein member of the TIM family that may play important roles in innate and adaptive immune responses. The overexpression of proteins associated with membrane proteins is a major obstacle to overcome in studies of membrane protein structures and functions. In this study, we successfully coupled the overexpression of the TIM-1 protein with a C-terminal enhanced green fluorescent protein (GFP) tag in Escherichia coli. To the best of our knowledge, this report is the first to describe the overexpression of human TIM-1 in E. coli. The purified TIM-1-EGFP fusion protein recognized and bound directly to apoptotic cells and did not to bind to viable cells. Furthermore, we confirmed that the interactions of TIM-1-EGFP with apoptotic cells were blocked by TIM-1-Fc fusion proteins. This fusion protein represents a readily obtainable source of biologically active TIM-1 that may prove useful in future studies of human TIM-1.