Clean and folded: Production of active, high quality recombinant human interferon-λ1

Abstract

Type III interferons exhibit antiviral activity against influenza viruses, coronaviruses, rotaviruses, and others. In addition, this type of interferon theoretically has therapeutic advantages, in comparison with type I interferons, due to its ability to activate a narrower group of genes in a relatively small group of target cells. Hence, it can elicit more targeted antiviral or immunomodulatory responses. Obtaining biologically-active interferon lambda (hIFN-λ₁) is fraught with difficulties at the stage of expression in soluble form or, in the case of expression in the form of inclusion bodies, at the stage of refolding. In this work, hIFN-λ₁ was expressed in the form of inclusion bodies, and a simple, effective refolding method was developed. Efficient and scalable methods for chromatographic purification of recombinant hIFN-λ₁ were also developed. High-yield, high-purity product was obtained through optimization of several processes including: recombinant protein expression; metal affinity chromatography; cation exchange chromatography; and an intermediate protein refolding stage. The obtained protein was shown to feature expected specific biological activity in line with published effects: induction of MxA gene expression in A549 cells and antiviral activity against influenza A virus.

Keywords: Cation exchange chromatography; Immobilized metal affinity chromatography; Interferon-lambda; Refolding.

Graphical abstract

Fig. 1

Recombinant hIFN-λ₁ protein construction. (a) Specifics of the pET302+ hIFN-λ₁ expression plasmid. (b) Amino acid sequence of the 6xHis-hIFN-λ₁ recombinant protein. Additional aa residues between the polyhistidine tag and hIFN-λ₁ are indicated in italics. Sequence regions identified using mass spectrometry (Section 3.4.1) are highlighted: black bars and white bars indicate enzymatic digestion with trypsin or chymotrypsin, respectively.

Fig. 2

Electrophoresis of lysate fractions from the hIFN-λ₁ producing strain (E. coli BL-21). (a) Induction testing. IPTG concentrations used for induction are highlighted (0.5 mM, 0.1 mM, 0.01 mM). Incubation temperatures are shown (left 37℃, right 32℃). SF – soluble fraction; IB – inclusion bodies. (b) Protein production. 'Overnight culture' - overnight culture, subcultured for production; 'Before induction' - cell lysate before addition of inducer; and 'After induction' - cell lysate, 3 h after addition of inducer. NC – negative control (E. coli BL-21 without plasmid).

Fig. 3

Purification stages of hIFN-λ₁. (a) Primary purification of recombinant hIFN-λ₁ by IMAC. Solid line: absorbance at 280 nm; the initial point of elution is marked with arrow. (b) Final purification of recombinant hIFN-λ₁ by cation exchange chromatography at room temperature (solid red line) and at +6 °C (solid black line). Solid lines indicate absorption at 280 nm (left y-axis); dashed line indicates conductivity of the solution (right y-axis). (c) PAGE of 6His-hIFN-λ₁-containing samples at various stages. Key: M – marker (Precision Plus mol. weight standard); ‘IMAC target’ – target peak from metal-saffinity column; ‘Refolding’ – material after refolding; ‘IEC’ – material after cation exchange chromatography (final stage); ‘SH’ – reducing PAGE conditions (with β-mercaptoethanol), ‘S-S’ – non-reducing conditions (without β-mercaptoethanol). Coomassie staining. (d) Gel filtration analytical chromatography of recombinant hIFN-λ₁. Solid line: absorbance at 280 nm. Peaks corresponding to hIFN-λ₁ monomers (∼98 %) and dimers (<1.5 %) are marked with arrows.

Fig. 4

Comparison of ELISA signal from recombinant hIFN-λ₁ stored in different conditions. In addition to the two storage conditions (+4 °C and -80 °C), a commercial standard is shown from the 'Human IL-29/IL-28B kit (IFN-lambda 1/3) DuoSet' ELISA kit (DY1598B, R&D Systems).

Fig. 5

Biological activity of recombinant hIFN-λ₁. (a) Change in MxA expression in A549 cells treated with hIFN-λ₁ (10 ng/mL) for 10 or 24 h during incubation. The y-axis shows relative MxA expression; the x-axis shows cell treatment conditions. The differences in MxA-2 mRNA expression between the control group at 10 h and the control group at 24 h were insignificant (p = 0.3333). T-test was performed using the nonparametric Mann-Whitney test. * p-value = 0.0286; ** p-value = 0.0159. (b) EC₅₀ determination (hIFN-λ₁ and influenza virus A/California/04/09 (H1N1)). The y-axes show the relative level of influenza nucleoprotein (100 % corresponds to the NP concentration in infected cells without hIFN-λ₁ pretreatment). The x-axes show hIFN-λ₁ concentration (ng/mL). The approximation was performed using a dose-response relationship with R² values of 0.97 and 0.98, for 0.004 moi and 0.016 moi, respectively.