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 p<smlcap>M</smlcap>. Our results demonstrate that using an MBP-tagged fusion is an efficient way to produce pure IFNα-2b.

Production of bioactive liver-targeting interferon Mu-IFN-CSP by soluble prokaryotic expression

“…After induction conditions optimized, the solubility of mu-IFN-CSP was up to 98.4%. The most optimal condition was at OD 600 = 0.9, inducing the culture at 34 °C for 6 h with 0.1 mM IPTG, which is consistent with previous reports that low temperature and low IPTG concentration may improve the solubility of target protein (Vu et al 2016 ; Xu et al 2006 ).…”

“…Most of them have reported that the IFNs are expressed as inclusion bodies in recombinant E. coli (Neves et al 2004 ; Srivastava et al 2005 ; Valente et al 2004 ). The refolding of misfolding and aggregation IFNs from inclusion bodies usually need extensive optimization, which makes the refolding procedures cumbersome (Middelberg 2002 ; Vu et al 2016 ). Up to now, it is still hard to produce soluble IFNs in recombinant E. coli .…”

“…Interferon (IFN) is a kind of cytokines with the ability to induce antiviral, immunomodulatory and anti-tumor effects (Degasperi et al 2017 ; Kotredes et al 2017 ; Vu et al 2016 ). IFNs have been applied as therapeutic medicine since first cloning and expressing interferon using DNA recombinant technology (Goeddel et al 1980 ).…”

Prokaryotic soluble expression and purification of bioactive human fibroblast growth factor 21 using maltose-binding protein

“…To this end, one of eight tags – His6, Sumo, Trx, GST, PDIb′a′, MBP, NusA, or PDI – was attached to the N terminus of hFGF21, resulting in eight recombinant vectors. Previously, these tags have been used in multiple studies to improve the soluble expression of various recombinant proteins in E. coli 19 – 23 , 27 , 28 . In the present study, the hFGF21 fusion vectors were transformed into the E. coli BL21(DE3) to characterise the behaviour of the fusion proteins expressed in the cytoplasm.…”

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“…N utilization substance protein A (NusA) is one of the most commonly used protein tagging labels for enhancing the yield and solubility of recombinant proteins (Cabrita et al, 2006;Vu et al, 2016;Sun et al, 2012). A fusion-tag efficiency evaluating assay indicated that seven out of eight tested large molecular proteins had been efficiently expressed at high solubility by NusA-tag, while only four were expressed with other tags such as thioredoxin (TRX), small ubiquitin-like modifier (SUMO), maltose-binding protein (MBP), glutathione S-transferase (GST) etc.…”