Expression and purification of MERS-CoV envelope protein, an essential viroporin, using the baculovirus expression system

Abstract

Background and objectives: The causative agent of Middle East Respiratory Syndrome (MERS) is a zoonotic Coronavirus (MERS-CoV) identified in Saudi Arabia in 2012. The envelope (E) protein of MERS-CoV is a small viral protein which plays several essential roles during virus replication. To facilitate study of the structure and function of the E protein, recombinant MERS-CoV E protein was expressed using the baculovirus expression system.

Materials and methods: A recombinant E open reading frame including an 8-histidine tag at the amino terminus was designed and cloned into a baculovirus transfer vector. Following construction of a recombinant virus insect cells were infected and the expression of the E protein assessed by SDS-PAGE and Western blotting.

Results: Recombinant E protein, tagged at the N-terminus with a polyhistidine sequence, with a molecular mass of 10.18 kD was identified by Western blotting with an anti-His antibody. Following large scale infection E protein was released by detergent mediated lysis of infected cells and purified by Immobilized Metal Ion Affinity Chromatography (IMAC).

Conclusion: Purified full length recombinant MERS-CoV E protein can be isolated by IMAC and is suitable for further functional, biophysical or immunological studies.

Keywords: Baculovirus; Coronaviruses; Envelope protein; Immobilised metal-affinity chromatography; Insect cells; MERS-CoV; Middle east respiratory syndrome.

Fig. 1

A-Schematic representation of the pTrix1.1 vector containing the MERS-CoV-E construct. Key features of the plasmid are marked. B-The MERS-CoV E protein sequence. The single predicted transmembrane region is underlined and the cysteines indicated.

Fig. 2

Recombinant MERS-E protein expression in insect cells revealed by Western blot. A-Lane 1: See Blue™ Plus 2 Pre-Stained Protein Standard (Invitrogen), lane 2 and 3: Infection by two recombinant E viruses, lane 4: negative control (mock infected cells). The arrow indicates a molecular mass of ∼10 kDa.

Fig. 3

MERS-CoV-E protein elution profile following IMAC affinity chromatography. The load is shown up to 100 min and the elution peak begins at fraction 3 – and extends over the subsequent 12 fractions. Fractions were of 2.5 min duration at a flow rate of 2 ml min⁻¹ (5mls).

Fig. 4

Analysis of the elution profile for recombinant MERS-CoV-E following expression in Sf9 cells and IMAC purification. A. Silver stained SDS-PAGE. B. Western Blot with anti-His antibody. The fractions are indicated. Markers to the left of each panel are in kilodaltons.

Fig. 5

Protein concentration assessment of MERS-CoV-E by SDS-PAGE next to a BSA standard. Marker is in kilodaltons (left of the gel). Lanes 1–3 represent 0.5, 1, 2.5 μg of BSA respectively. The arrows indicate a molecular mass of ∼66.5 and 10.18 kDa for BSA and MERS-CoV-envelope protein respectively.