High yield soluble bacterial expression and streamlined purification of recombinant human interferon α-2a

Abstract

Interferon α-2a (IFNA2) is a member of the Type I interferon cytokine family, known for its antiviral and anti-proliferative functions. The role of this family in the innate immune response makes it an attractive candidate for the treatment of many viral and chronic immune-compromised diseases. Recombinant IFNA2 is clinically used to modulate hairy cell leukemia as well as hepatitis c. Historically, IFNA2 has been purified from human leukocytes as well as bacterial expression systems. In most cases, bacterial expression of IFNA2 resulted in inclusion body formation, or required numerous purification steps that decreased the protein yield. Here, we describe an expression and purification scheme for IFNA2 using a pET-SUMO bacterial expression system and a single purification step. Using SUMO protein as the fusion tag achieved high soluble protein expression. The SUMO tag was cleaved with the Ulp1 protease leaving no additional amino acids on the fusion terminus following the cleavage. Mass spectrometry, circular dichroism, 2D heteronuclear NMR, and analytical ultracentrifugation confirmed the amino acid sequence identity, secondary and tertiary protein structures, and the solution behavior of the purified IFNA2. The purified protein also has antiviral and anti-proliferative activities comparable to the WHO International Standard, NIBSC 95/650, and the IFNA2 standard available from PBL Assay Science. Combining the expression and purification protocols developed here to produce IFNA2 on a laboratory scale with the commercial fermenter technology commonly used in pharmaceutical industry may further enhance IFNA2 yields, which will promote the development of interferon-based protein drugs to treat various disorders.

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

“…During the purification process, auto-cleavage of the fusion protein can occur, resulting in cleaved MBP and IFNα-2b. The same finding can be observed in other publications, for example during the purification of a GST-IFNα-2b fusion construct [Rabhi-Essafi et al, 2007] and a SUMO fusion construct [Bis et al, 2014]. One way to reduce auto-cleavage is to add PMSF during the purification procedure, especially before the protease cleavage step.…”

“…Our use of the anion exchange chromatography as the final step in the purification process successfully removes endotoxins. Recently, for example, the SUMO tag has been recruited to produce 16 mg of IFNA2 per liter of cell culture [Bis et al, 2014], which is similar to our final yield of 14.4 mg/l of cell culture.…”

“…This ambiguity does not arise when using the ISRE promoter-based luciferase assay to test IFN activity. Previously published studies have used antiviral and antiproliferative assays to determine the biological activity [Bis et al, 2014;Schmeisser et al, 2006]; however, these methods are not optimal because they are affected by contaminant LPS from protein production [Malcolm and Worthen, 2003;Nolan et al, 2003]. Our method for testing the biological activity using a firefly luciferase assay cloned with an ISRE promoter is a more accurate method for determining in vitro IFNα-2b activity because it is resistant to the effects of endotoxins.…”

“…Our method for testing the biological activity using a firefly luciferase assay cloned with an ISRE promoter is a more accurate method for determining in vitro IFNα-2b activity because it is resistant to the effects of endotoxins. Furthermore, the purification in these papers [Bis et al, 2014;Schmeisser et al, 2006] stopped after the second IMAC step after tag cleavage. In our experience, the purity of the IFNα-2b at this step is approximately only 88% (online suppl.…”

Expression and purification of Canis interferon α in Escherichia coli using different tags

“…For example, Yang expressed feline interferon both in E. coli and P. pastoris with no significant difference in anti-viral activity [26]. Bis expressed human IFNα in E. coli and purified it to homogeneity in a single step [27]. Srivastava overexpressed human IFNα in E. coli and achieved high yields [28].…”