Soluble Prokaryotic Expression and Purification of Human Interferon Alpha-2b Using a Maltose-Binding Protein Tag

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 IFN α-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 IFN α-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 EC < sub > 50 < /sub > of 10.3 ± 5.9 p < smlcap > M < /smlcap > . Our results demonstrate that using an MBP-tagged fusion is an efficient way to produce pure IFN α-2b. < br / > J Mol Microbiol Biotechnol 2016;26:359-368
Source: Journal of Molecular Microbiology and Biotechnology - Category: Microbiology Source Type: research