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Article ; Online: Micellization-induced amplified fluorescence response for highly sensitive detection of heparin in serum

Yeon Jin Jang / Boyun Kim / Euijin Roh / Hyunuk Kim / Seoung Ho Lee

Scientific Reports, Vol 10, Iss 1, Pp 1-

2020 Volume 9

Abstract: Abstract Fluorescence-based assays should be feasible in aqueous media for effectively detecting the biological factors. However, numerous sensors have limited signal transductions and low fluorescence quantum yields due to the ingerently reduced excited ...

Abstract	Abstract Fluorescence-based assays should be feasible in aqueous media for effectively detecting the biological factors. However, numerous sensors have limited signal transductions and low fluorescence quantum yields due to the ingerently reduced excited state energy of fluorophores in aqueous solution, which reduces their sensitivity. This necessitates a smart sensing approach with an amplified fluorescence response for analytes in aqueous solution. Herein, a new building block which self-assembles in aqueous media, giving a micellar sturcuture with the hydrophobic π-extended conjugated system at the core and hydrophilic groups at the periphery, was devised for the first time. We demonstrated that the aggregated fluorophores in a micelle induce amplified fluorescence quenching, in which the excited electron efficiently migrates through π-extended conjugated system in a micelle, as in a polymeric system. Such feature differentiates this sensing approach from the numerous fluorescence-based tools previously developed for sensitive detection. This new system exhibited highly sensitive signal transduction for specific analytes even under actual bioanalytical conditions.
Keywords	Medicine ; R ; Science ; Q
Subject code	500
Language	English
Publishing date	2020-06-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Soluble Cytoplasmic Expression and Purification of Immunotoxin HER2(scFv)-PE24B as a Maltose Binding Protein Fusion

Sangsu Park / Minh Quan Nguyen / Huynh Kim Khanh Ta / Minh Tan Nguyen / Gunsup Lee / Chong Jai Kim / Yeon Jin Jang / Han Choe

International Journal of Molecular Sciences, Vol 22, Iss 6483, p

2021 Volume 6483

Abstract: Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial ... ...

Abstract	Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa , consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli , and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli . Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC 50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM ( n = 9) and 19 ± 1.4 pM ( n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.
Keywords	protein expression ; protein purification ; immunotoxin ; HER2(scFv)-PE24B ; maltose binding protein ; trastuzumab ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	572
Language	English
Publishing date	2021-06-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: DJ-1 Can Replace FGF-2 for Long-Term Culture of Human Pluripotent Stem Cells in Defined Media and Feeder-Free Condition

Julee Kim / Sangki Baek / Yean-Ju Hong / Michelle Novais de Paula / Musharrat Jahan Prima / Yeon-Mok Oh / Sun-Shin Cha / Jeong-Tae Do / Yeon-Jin Jang / Han Choe

International Journal of Molecular Sciences, Vol 22, Iss 5954, p

2021 Volume 5954

Abstract: Conventional human pluripotent stem cell (hPSC) cultures require high concentrations of expensive human fibroblast growth factor 2 (hFGF-2) for hPSC self-renewal and pluripotency in defined media for long-term culture. The thermal instability of the hFGF- ...

Abstract	Conventional human pluripotent stem cell (hPSC) cultures require high concentrations of expensive human fibroblast growth factor 2 (hFGF-2) for hPSC self-renewal and pluripotency in defined media for long-term culture. The thermal instability of the hFGF-2 mandates media change every day, which makes hPSC culture costly and cumbersome. Human DJ-1 (hDJ-1) can bind to and stimulate FGF receptor-1. In this study, for the first time, we have replaced hFGF-2 with hDJ-1 in the essential eight media and maintained the human embryonic stem cells (hESCs), H9, in the defined media at feeder-free condition. After more than ten passages, H9 in both groups still successfully maintained the typical hESC morphology and high protein levels of pluripotency markers, SSEA4, Tra1-60, Oct4, Nanog, and ALP. DNA microarray revealed that more than 97% of the 21,448 tested genes, including the pluripotency markers, Sox2, Nanog, Klf4, Lin28A, Lin28B, and Myc, have similar mRNA levels between the two groups. Karyotyping revealed no chromosome abnormalities in both groups. They also differentiated sufficiently into three germ layers by forming in vitro EBs and in vivo teratomas. There were some variations in the RT-qPCR assay of several pluripotency markers. The proliferation rates and the mitochondria of both groups were also different. Taken together, we conclude that hDJ-1 can replace hFGF-2 in maintaining the self-renewal and the pluripotency of hESCs in feeder-free conditions.
Keywords	Keywords: hPSC ; DJ-1 ; FGF-2 ; Defined media ; Feeder-free ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	070
Language	English
Publishing date	2021-05-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b′a′ Domain

Thi Kieu Oanh Nguyen / Thi Luong Vu / Minh Quan Nguyen / Huynh Kim Khanh Ta / Kyoung Sun Park / Soo Hyeon Kim / Chong Jai Kim / Yeon Jin Jang / Han Choe

International Journal of Molecular Sciences, Vol 22, Iss 5267, p

2021 Volume 5267

Abstract: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as ... ...

Abstract	Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b′a′ domain of protein disulfide isomerase (PDIb′a′) greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb′a′-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC 50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.
Keywords	hGM-CSF ; MBP ; PDI ; ClearColi ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	616
Language	English
Publishing date	2021-05-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Novel Bacterial Production of Two Different Bioactive Forms of Human Stem-Cell Factor

Eunyoung Lee / Michelle Novais de Paula / Sangki Baek / Huynh Kim Khanh Ta / Minh Tan Nguyen / Taeck-Hyun Jeong / Chong Jai Kim / Yeon Jin Jang / Han Choe

International Journal of Molecular Sciences, Vol 22, Iss 6361, p

2021 Volume 6361

Abstract: Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three ... ...

Abstract	Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N -terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b’a’ domain (PDIb’a’) tags were attached to the N -terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC 50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.
Keywords	human stem-cell factor ; hSCF248 ; hSCF164 ; MBP ; recombinant protein ; soluble protein ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	500
Language	English
Publishing date	2021-06-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Antinociceptive and Anti-Inflammatory Effects of Recombinant Crotamine in Mouse Models of Pain

Jong Yeon Park / Bich Hang Do / Ju-Seung Lee / Hyun Cheol Yang / Anh Ngoc Nguyen / Martin Krupa / Chong Jai Kim / Yeon Jin Jang / Han Choe

Toxins, Vol 13, Iss 707, p

2021 Volume 707

Abstract: Crotamine, a toxin found in the venom of the South American rattlesnake Crotalus durissus terrificus , has been reported to have antinociceptive effects. We purified recombinant crotamine expressed in Escherichia coli and investigated its antinociceptive ...

Abstract	Crotamine, a toxin found in the venom of the South American rattlesnake Crotalus durissus terrificus , has been reported to have antinociceptive effects. We purified recombinant crotamine expressed in Escherichia coli and investigated its antinociceptive and anti-inflammatory effects using the hot-plate test, acetic-acid-induced writhing method, and formalin test in mice. Recombinant crotamine was administered intraperitoneally (0.04–1.2 mg kg −1 ) or intraplantarly (0.9–7.5 μg 10 μL −1 ) before the tests. The paw volume was measured with a plethysmometer. To evaluate the antagonistic and anti-inflammatory effects of naloxone, subcutaneous naloxone (4 mg kg −1 ) or intraplantar naloxone (5 μg 10 μL −1 ) was administered before recombinant crotamine. For tumor necrosis factor (TNF)-α assays, blood was drawn 3 h after formalin injection and measured using enzyme-linked immunosorbent assay. Intraperitoneal and intraplantar recombinant crotamine had antinociceptive and anti-inflammatory effects, neither of which were affected by pre-treatment with naloxone. The mean serum TNF-α levels were significantly lower in the intraperitoneal recombinant crotamine (0.4 and 1.2 mg kg −1 ) or intraplantar (2.5 and 7.5 μg 10 μL −1 ) recombinant crotamine groups than in the saline group and were not affected by naloxone pre-treatment. In conclusion, recombinant crotamine possesses significant antinociceptive and anti-inflammatory effects that do not appear to be related to the opioid receptor. The antinociceptive and anti-inflammatory effects of intraperitoneal or intraplantar recombinant crotamine are related to TNF-α.
Keywords	crotamine ; nociception ; inflammation ; naloxone ; tumor necrosis factor-α ; Medicine ; R
Subject code	333
Language	English
Publishing date	2021-10-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Enhanced ANGPTL2 expression in adipose tissues and its association with insulin resistance in obese women

Jimin Kim / Seul Ki Lee / Yeon Jin Jang / Hye Soon Park / Jong-Hyeok Kim / Joon Pio Hong / Yeon Ji Lee / Yoon-Suk Heo

Scientific Reports, Vol 8, Iss 1, Pp 1-

2018 Volume 12

Abstract: Abstract Angiopoietin-like protein 2 has been proposed to be a key mediator linking obesity and insulin resistance. However, no detailed study of ANGPTL2 expression in human adipose tissues has yet been reported. To investigate the pattern and regulation ...

Abstract	Abstract Angiopoietin-like protein 2 has been proposed to be a key mediator linking obesity and insulin resistance. However, no detailed study of ANGPTL2 expression in human adipose tissues has yet been reported. To investigate the pattern and regulation of ANGPTL2 expression in human adipose tissues in obesity and its related diseases, we recruited 32 non-diabetic and 13 type 2 diabetic obese women and 32 normal-weight women. ANGPTL2 mRNA was expressed at a similar level in visceral and subcutaneous adipose tissues. Adipose tissue ANGPTL2 mRNA was much higher in obese patients. Adipose tissue ANGPTL2 mRNA and serum ANGPTL2 levels showed strong associations with metabolic parameters associated with insulin resistance. In adipose tissue, ANGPTL2 mRNA was closely correlated with the expression of genes involved in inflammation and ER stress. ANGPTL2 mRNA was principally expressed in adipocytes, and its expression was markedly higher in the adipocyte but non-adipocyte fraction of obese adipose tissues. Culture of human adipocytes under conditions mimicking the microenvironment of obese adipose tissue (especially, increased ER stress) stimulated ANGPTL2 gene expression and secretion. In addition, co-culture of adipocytes and macrophages suggested that ANGPTL2 excessively produced by adipocytes, may contribute inflammation and remodeling in obese adipose tissues, thereby promoting insulin resistance.
Keywords	Angptl4 Expression ; Angptl4 mRNA ; Obese Adipose Tissue ; Angiopoietin-like Protein ; MMP-9 mRNA ; Medicine ; R ; Science ; Q
Subject code	610
Language	English
Publishing date	2018-09-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Prokaryotic soluble overexpression and purification of oncostatin M using a fusion approach and genetically engineered E. coli strains

Minh Tan Nguyen / Musharrat Jahan Prima / Jung-A. Song / Julee Kim / Bich Hang Do / Jiwon Yoo / Sangsu Park / Jaepyeong Jang / Sunju Lee / Eunyoung Lee / Michelle de Paula Novais / Hyeon-Beom Seo / Seon-yeong Lee / Mi-La Cho / Chong Jai Kim / Yeon Jin Jang / Han Choe

Scientific Reports, Vol 9, Iss 1, Pp 1-

2019 Volume 13

Abstract: Abstract Human Oncostatin M (OSM), initially discovered as a tumour inhibitory factor secreted from U-937 cells, is a gp130 (IL-6/LIF) cytokine family member that exhibits pleiotropic effects in inflammation, haematopoiesis, skeletal tissue alteration, ... ...

Abstract	Abstract Human Oncostatin M (OSM), initially discovered as a tumour inhibitory factor secreted from U-937 cells, is a gp130 (IL-6/LIF) cytokine family member that exhibits pleiotropic effects in inflammation, haematopoiesis, skeletal tissue alteration, liver regeneration, cardiovascular and metabolic diseases. Cytoplasmic expression of OSM in Escherichia coli results in inclusion bodies, and complex solubilisation, refolding and purification is required to prepare bioactive protein. Herein, eight N-terminal fusion variants of OSM with hexahistidine (His6) tag and seven solubility-enhancing tags, including thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (Nusa), human protein disulphide isomerase (PDI) and the b‘a’ domain of PDI (PDIb‘a’), were tested for soluble OSM expression in E. coli. The His6-OSM plasmid was also introduced into genetically engineered Origami 2 and SHuffle strains to test expression of the protein. At 18 °C, MBP-tagged OSM was highly expressed and solubility was dramatically enhanced. In addition, His6-OSM was more highly expressed and soluble in Origami 2 and SHuffle strains than in BL21(DE3). MBP-OSM and His6-OSM were purified more than 95% with yields of 11.02 mg and 3.27 mg from a 500 mL culture. Protein identity was confirmed by mass spectroscopy, and bioactivity was demonstrated by in vitro inhibition of Th17 cell differentiation.
Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2019-09-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Correction

Jung-A Song / Bon-Kyung Koo / Seon-Ha Chong / Kyunhoo Kim / Dong Kyu Choi / Thu Trang Thi Vu / Minh Tan Nguyen / Boram Jeong / Han-Bong Ryu / Injune Kim / Yeon Jin Jang / Robert Charles Robinson / Han Choe

PLoS ONE, Vol 9, Iss

Soluble Expression of Human Leukemia Inhibitory Factor with Protein Disulfide Isomerase in Escherichia coli and Its Simple Purification

2014 Volume 1

Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2014-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Correction

PLoS ONE, Vol 9, Iss

Soluble Expression of Human Leukemia Inhibitory Factor with Protein Disulfide Isomerase in and Its Simple Purification.

2014 Volume 1

Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2014-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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