title: Soluble Prokaryotic Expression and Purification of Human Interferon Alpha-2b Using a Maltose-Binding Protein Tag.
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.
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea.
authors.forename: Thu Trang Thi
authors.initials: TT
authors.lastname: Vu
authors.forename: Boram
authors.initials: B
authors.lastname: Jeong
authors.forename: Martin
authors.initials: M
authors.lastname: Krupa
authors.forename: Uijung
authors.initials: U
authors.lastname: Kwon
authors.forename: Jung-A
authors.initials: JA
authors.lastname: Song
authors.forename: Bich Hang
authors.initials: BH
authors.lastname: Do
authors.forename: Minh Tan
authors.initials: MT
authors.lastname: Nguyen
authors.forename: Taewook
authors.initials: T
authors.lastname: Seo
authors.forename: Anh Ngoc
authors.initials: AN
authors.lastname: Nguyen
authors.forename: Chul Hyun
authors.initials: CH
authors.lastname: Joo
authors.forename: Han
authors.initials: H
authors.lastname: Choe
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D058619:Maltose-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D000077190:Interferon alpha-2; D016898:Interferon-alpha; C000614170:Interferon-alpha2b; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins