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  1. 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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  2. Article ; Online: Granulocyte colony-stimulating factor (GCSF) fused with Fc Domain produced from E. coli is less effective than Polyethylene Glycol-conjugated GCSF

    Bich Hang Do / Hyo Jeong Kang / Jung-A Song / Minh Tan Nguyen / Sangsu Park / Jiwon Yoo / Anh Ngoc Nguyen / Grace G. Kwon / Jaepyeong Jang / Mihee Jang / Sunju Lee / Seoungjun So / Seongrak Sim / Kyung Jin Lee / Mark J. Osborn / Han Choe

    Scientific Reports, Vol 7, Iss 1, Pp 1-

    2017  Volume 9

    Abstract: Abstract Human granulocyte colony-stimulating factor (GCSF) is a well-known cytokine for neutropenia treatment. However, daily injections are required due to the short circulating half-life of the protein. To overcome this bottleneck, we fused GCSF with ... ...

    Abstract Abstract Human granulocyte colony-stimulating factor (GCSF) is a well-known cytokine for neutropenia treatment. However, daily injections are required due to the short circulating half-life of the protein. To overcome this bottleneck, we fused GCSF with the Fc domain of IgG1 at the C terminus (GCSF-Fc) and with the maltose binding protein (MBP) tag at the N-terminus and expressed it as a soluble protein in the cytoplasm of E. coli. We also conjugated PEG aldehyde to GCSF to make PEG-GCSF. The bioactivities of GCSF-Fc and PEG-GCSF were similar to native GCSF using the mouse M-NFS-60 myelogenous leukemia cell line. The EC50 dose-response curves for GCSF, GCSF-Fc and PEG-GCSF were 37 ± 12 pM, 75 ± 13.5 pM and 46 ± 5.5 pM, respectively. When the proteins were injected into neutropenic rats, the group injected with PEG-GCSF showed the highest and fastest recovery of neutrophils, followed by GCSF-Fc and GCSF. ELISA assay revealed the PEG-GCSF had the longest plasma circulation (>72 h), followed by GCSF-Fc (>48 h) and GCSF (~24 h), which is consistent with the in vivo activities of the proteins. In summary, the GCSF-Fc purified from E. coli was not as efficient as PEG-GCSF in treating neutropenic rats.
    Keywords Medicine ; R ; Science ; Q
    Language English
    Publishing date 2017-07-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article: Soluble expression and purification of bioactive interleukin 33 in E. coli

    Do, BichHang / Sangsu Park / Grace G. Kwon / Minh Tan Nguyen / Hyo Jeong Kang / Jung-A Song / Jiwon Yoo / Anh Ngoc Nguyen / Jaepyeong Jang / Mihee Jang / Sunju Lee / Seoungjun So / Sungrak Sim / Jonghwa Jin / Kyung Jin Lee / Mark J. Osborn / Han Choe

    Biotechnology and bioprocess engineering. 2017 June, v. 22, no. 3

    2017  

    Abstract: Interleukin-33 (IL-33) is one of the important alarmins of the immune system and possesses dual functions as an anti- or pro-inflammatory molecule. The production of this cytokine in E. coli is hampered by the insoluble expression in the cytoplasm, ... ...

    Abstract Interleukin-33 (IL-33) is one of the important alarmins of the immune system and possesses dual functions as an anti- or pro-inflammatory molecule. The production of this cytokine in E. coli is hampered by the insoluble expression in the cytoplasm, resulting in inclusion body formation. In this study, the expression of IL-33 was optimized by fusing the N-terminus of IL-33 with several solubilizing tags that act as chaperones for proper protein folding: maltose binding protein (MBP), b´a´ domain of protein disulfide isomerase (PDIb´a´) and glutathione Stransferase (GST). The expression of the fusion proteins was stimulated by 0.5 mM IPTG at different temperatures, 37, 30, 25, and 18°C. As a result, IL-33 was expressed highly and in soluble form in the cytoplasm of E. coli when fused with MBP or PDIb´a´ tags in the presence of 0.5 mM IPTG at 25 or 30°C. We describe a simple purification procedure of IL-33 from the PDIb´a´-IL-33 construct using immobilized metal affinity chromatography (IMACs) with supplementary of tobacco etch virus (TEV) protease for tag removal. The high bioactivity of purified IL-33 on the proliferation and activation of macrophages was confirmed by MTT and nitrite releasing assays using RAW 264.7 These data show an improved method for producing high grade and yield IL-33.
    Keywords Escherichia coli ; Tobacco etch virus ; affinity chromatography ; binding proteins ; bioactive properties ; cytoplasm ; glutathione ; interleukins ; macrophages ; maltose ; nitrites ; protein disulfide-isomerase ; protein folding ; proteinases ; solubilization ; temperature
    Language English
    Dates of publication 2017-06
    Size p. 256-264.
    Publishing place The Korean Society for Biotechnology and Bioengineering
    Document type Article
    ZDB-ID 2125481-3
    ISSN 1976-3816 ; 1226-8372
    ISSN (online) 1976-3816
    ISSN 1226-8372
    DOI 10.1007/s12257-017-0060-0
    Database NAL-Catalogue (AGRICOLA)

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