1. Soluble Prokaryotic Expression and Purification of Human Interferon Alpha-2b Using a Maltose-Binding Protein Tag.
- Author
-
Vu TT, Jeong B, Krupa M, Kwon U, Song JA, Do BH, Nguyen MT, Seo T, Nguyen AN, Joo CH, and Choe H
- Subjects
- Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Humans, Interferon alpha-2, Interferon-alpha genetics, Maltose-Binding Proteins genetics, Recombinant Fusion Proteins genetics, Chromatography, Affinity methods, Interferon-alpha isolation & purification, Interferon-alpha metabolism, Maltose-Binding Proteins isolation & purification, Maltose-Binding Proteins metabolism, Recombinant Fusion Proteins isolation & purification, Recombinant Fusion Proteins metabolism
- 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 chromatography, 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 EC50 of 10.3 ± 5.9 pM. Our results demonstrate that using an MBP-tagged fusion is an efficient way to produce pure IFNα-2b., (© 2016 S. Karger AG, Basel.)
- Published
- 2016
- Full Text
- View/download PDF