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 the 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 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 had 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.

Keywords: Bacterial expression; Interferon α-2a; Purification; SUMO; Soluble protein.

Figure 1

Post-translation fusion complex and the corresponding protein sequence. A) Pictorial representation of the translated His₆-SUMO-IFNA2 fusion complex with the Ulp1 cleavage site indicated by an arrow. B) The primary protein sequence of IFNA2 following cleavage of the fusion tag from IFNA2, which matches with that of the pharmaceutical product as well as the protein for which the NMR structure has been previously determined (PDB ID 1ITF).

Figure 2

Schematic of expression and purification scheme optimized to obtain high yields of highly pure, soluble IFNA2 from E. coli.

Figure 3

Purification of the His₆-SUMO-IFNA2 fusion complex. The lysate fraction was loaded onto a nickel column and the flow-through was collected. A low concentration imidazole wash allowed for further removal of any bacterial contaminants. Eluates 2–5 show the fusion complex eluting from the nickel resin, with overexpression of the complex evident in Eluate 3 and Eluate 4 at ~33kDa. Molecular weight markers are shown on the far left.

Figure 4

Cleavage of the His₆-SUMO tag with Ulp1 protease. Pre-treatment refers to elution fractions from Figure 3 prior to addition of Ulp1 protease. Following the cleavage reaction, the post-treatment lane shows two distinct bands representing the His₆-SUMO tag and untagged IFNA2. Pure IFNA2 was collected in the flow-through. A high concentration imidazole elution fraction removes the His₆-SUMO tag from the nickel column.

Figure 5

MS analysis of IFNA2. A) Intact full-length protein injection shows a predominant peak at 19237 Da. B) Tryptically digested IFNA2 initially showed a residual serine on the N-terminus of the protein, indicated by the 1457 m/z peak. The inset text shows the N-terminal fragment corresponding to this m/z value. The upper and lower panels show pre- and post-deletion of the N-terminal serine, respectively. C) Tryptically digested IFNA2 shows the presence of the 1526 m/z peak, corresponding to the N-terminal cysteine. The inset text shows the N-terminal fragment at this m/z value. The upper and lower panels show pre- and post-deletion of the N-terminal serine, respectively.

Figure 6

Biophysical characterization of IFNA2. A) Far-UV CD spectrum of IFNA2 shows that this protein is well-folded and α-helical. B) Sedimentation velocity AUC demonstrates that IFNA2 is a monomer in solution at pharmaceutically relevant concentrations.

Figure 7

2D HSQC NMR spectrum of IFNA2, and comparison with the previously published crosspeak assignments. The blue peaks correspond to IFNA2 data collected at pH 3.5. Overlaid numerical values correspond to peak assignments as determined by Klaus et al. [20].

Figure 8

Functional assays of IFNA2 in comparison with the two well-established standards. A) IFNA2 antiviral activity following encephalomyocarditis (EMC) viral challenge on human A549 cells. B) IFNA2 anti-proliferative function against human ovarian cancer cells. Red, blue, and green symbols represent the data on our purified IFNA2, E. coli-derived interferon α-2a standard from PBL Assay Science, and on the 2^nd International WHO standard for interferon α-2a (NIBSC 95/650), respectively. C) The EC50 values determined from data fitting.