Supplementary Material for: Soluble Prokaryotic Expression and Purification of Human Interferon Alpha-2b Using a Maltose-Binding Protein Tag Vu T.T.T. Jeong B. Krupa M. Kwon U. Song J.-A. Do B.H. Nguyen M.T. Seo T. Nguyen A.N. Joo C.H. Choe H. 10.6084/m9.figshare.3502823.v1 https://karger.figshare.com/articles/figure/Supplementary_Material_for_Soluble_Prokaryotic_Expression_and_Purification_of_Human_Interferon_Alpha-2b_Using_a_Maltose-Binding_Protein_Tag/3502823 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 <i>Escherichia coli</i> 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 <i>S</i>-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 <i>E. coli</i>. 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. 2016-07-27 11:16:07 Interferon alpha Maltose-binding protein Fusion protein Protein purification Soluble protein expression