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Soluble Prokaryotic Expression and Purification of Human Interferon Alpha-2b Using a Maltose-Binding Protein Tag.
- Source :
- Journal of Molecular Microbiology & Biotechnology; Oct2016, Vol. 26 Issue 6, p359-368, 10p
- Publication Year :
- 2016
-
Abstract
- Human interferon alpha-2b (IFNα-2b) has therapeutic applications as an antiviral and antiproliferative drug and has been used for a wide range of indications. Efficient production of IFNα-2b in Escherichia coli has been difficult because the protein tends to form inclusion bodies. This obstacle has garnered interest in efficiently expressing IFNα-2b and overcoming its poor solubility. In this study, seven N-terminal fusion partners - hexahistidine (His6), thioredoxin, glutathione S -transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A, protein disulfide bond isomerase (PDI), and b'a' domain of PDI - were tested for soluble overexpression of codon-optimized IFNα-2b in E. coli . Low temperature increased the expression level of all of the tagged proteins except for the GST fusion. All the tags, except for His6 and GST, improved solubility. We purified IFNα-2b from the MBP-tagged fusion using immobilized metal affinity chromatography and anion exchange chromatogra phy, and obtained a final yield of 7.2 mg from an initial 500-ml culture. The endotoxin level was 0.46 EU/μg. Biological activity was demonstrated using a luciferase assay, which showed a dose-dependent response with a calculated EC 50 of 10.3 ± 5.9 p M . Our results demonstrate that using an MBPtagged fusion is an efficient way to produce pure IFNα-2b. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 14641801
- Volume :
- 26
- Issue :
- 6
- Database :
- Complementary Index
- Journal :
- Journal of Molecular Microbiology & Biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 119119458
- Full Text :
- https://doi.org/10.1159/000446962