Abstract
Background: Full length Ebolavirus glycoprotein (GP) intersperses the outer most lipid membrane to form spikes, where it mediates virus-host cell interaction. A secretory form of GP (sGP) is also produced by all 5 known Ebolavirus species. These attributes make GP an ideal target for research and development (R and D) of Ebolavirus and possibly pan-filovirus targeted Rapid Diagnostic Tests (RDTs), bio-therapeutics and vaccines. Prior cloning of recombinant Zaire ebolavirus (EBOV) GP has majorly used insect (baculovirus ) expression systems. We report the cloning, expression and purification of the full length and extracellular domain (ECD) forms of recombinant EBOV GP in mammalian cell-lines. Methods and results: 2034 and 1956 base-pair (bp) coding DNA sequences corresponding to the 669 and 643 amino acids (aa) residues of full length and ECD forms of EBOV GP were sub-cloned into the pTGE plasmids. Recombinant pTGE-plasmids were used to transfect 293-6E HEK mammalian cells grown in serum-free FreeStyleTM 293 Expression Medium. Cell lysates and or culture supernatants were used to obtain purified protein, followed by analysis on SDS-PAGE and Western blot. Purified full length GP was detected as membrane bound protein in cell lysates with estimated molecular weight of ~100 kDa (Cal.M.W.~71.67 kDa) on Western blot; and 0.02 mg GP (Concentration: 0.2 mg/mL, Purity: ~50%) derived. On the contrary, ECD GP was detected in supernatants of cell culture broth with estimated molecular weights of ~116 kDa based on SDS-PAGE and Western blot; and 1.6 mg (Concentration: 0.4 mg/ml, Purity: ~70%) of GP_ECD was obtained. Conclusion: Within mammalian cells, recombinant full length EBOV GP is predominantly expressed as transmembrane protein (tGP), while ECD GP is eluted into the culture medium. Both recombinant forms of GP are critical for the R and D of rapid diagnostic tests (RDTs).
Highlights
Full length Ebolavirus glycoprotein (GP) intersperses the outer most lipid membrane to form spikes, where it mediates virus-host cell interaction
Whereas majority prior attempts to aimed to express recombinant GP have exploited insect expression systems, mammalian cell expressed recombinant forms of GP might be more representative of GP proteolytic and or post-translational modifications that occur in vivo, thereby making them more appropriate for R and D of EBOV targeted rapid diagnostic tests (RDTs), biotherapeutics and vaccines
Extracellular domain (ECD) GP was expressed in a secretory manner similar to what is observed during viral expression of the secretory form of GP (sGP) variant of EBOV
Summary
Ebolavirus is one of two common genera of the enveloped, nonsegmented single-stranded RNA virus family Filoviridae, order Mononegavirales [1]. Contrary to EBOV that requires transcriptional editing to express tGP, the GP gene of MARV is organized in a way that transcription results in a single sub-genomic RNA species used for the synthesis of the full-length envelope glycoprotein [13,14]. Through target-site mutation (truncation), the same cDNA was modified to generate the 1956 nucleotide bp coding DNA sequences corresponding to the 643 amino acids (aa) long residues of the extracellular domain (ECD) variant of EBOV GP (Figure 2) These were sub-cloned into the pTGE plasmids. This attempt to purify the target full length GP from cell culture supernatant was unsuccessful as very little protein could be detected in eluted fractions. The target protein GP_ECD was detected with estimated molecular weights of ~116 kDa based on SDS‐PAGE and Western blot analysis as shown in Figure 9. 1.6 mg (Concentration: 0.4 mg/ml, Purity: ~70%) of GP_ECD was obtained from 400 ml of cells at a density of 2–3 × 106 cells ml −1
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