Article ; Online: Prokaryotic soluble overexpression and purification of oncostatin M using a fusion approach and genetically engineered E. coli strains.
2019 Volume 9, Issue 1, Page(s) 13706
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 ... ...
| 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. |
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| MeSH term(s) | Escherichia coli ; Gene Expression ; Genetic Engineering ; Histidine ; Humans ; Maltose-Binding Proteins/metabolism ; Oligopeptides ; Oncostatin M/genetics ; Oncostatin M/metabolism ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/metabolism ; Solubility |
| Chemical Substances | His-His-His-His-His-His ; Maltose-Binding Proteins ; Oligopeptides ; Recombinant Fusion Proteins ; Oncostatin M (106956-32-5) ; Histidine (4QD397987E) |
| Language | English |
| Publishing date | 2019-09-23 |
| Publishing country | England |
| Document type | Journal Article ; Research Support, Non-U.S. Gov't |
| ZDB-ID | 2615211-3 |
| ISSN | 2045-2322 ; 2045-2322 |
| ISSN (online) | 2045-2322 |
| ISSN | 2045-2322 |
| DOI | 10.1038/s41598-019-50110-6 |
| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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