Filoviruses are highly virulent pathogens capable of causing severe disease. The glycoproteins of filoviruses are the only virally expressed proteins on the virion surface and are required for receptor binding. As such, they are the main candidate vaccine antigen. Despite their virulence, most filoviruses are not comprehensively characterized, and relatively few commercially produced reagents are available for their study. Here, we describe two methods for production and purification of filovirus glycoproteins in insect and mammalian cell lines. Considerations of expression vector choice, modifications to sequence, troubleshooting of purification method, and glycosylation differences are all important for successful expression of filovirus glycoproteins in cell lines. Given the scarcity of commercially available filovirus glycoproteins, we hope our experiences with possible difficulties in purification of the proteins will facilitate other researchers to produce and purify filovirus glycoproteins rapidly.
Filoviruses (mononegaviral family Filoviridae) are a group of highly virulent pathogens that can cause viral hemorrhagic fevers in humans and/or nonhuman primates. Currently, eight recognized filoviruses are classified into three genera: Cuevavirus, Ebolavirus, and Marburgvirus 1. The members of the genus Ebolavirus, i.e., ebolaviruses, are Bundibugyo (BDBV), Ebola virus (EBOV), Reston virus (RESTV), Sudan virus (SUDV), and Taï Forest virus (TAFV). All ebolaviruses except RESTV cause Ebola virus disease (EVD) in humans. The genus Marburgvirus, i.e., marburgviruses, contains Marburg virus (MARV) and Ravn virus (RAVV), both of which cause Marburg virus disease (MVD) in humans2. Finally, the genus Cuevavirus has a single member, Lloviu virus (LLOV), which has been associated with lethal disease in bats but has unknown pathogenicity for primates3.
Filovirions enter target cells through interaction of their only particle surface protein, glycoprotein GP1,2, with cell-surface attachment factors and Niemann Pick C1 (NPC1) as the common endosomal entry receptor4,5. GP1,2, is a typical class I fusion type 1 transmembrane protein that is highly glycosylated and serves as the primary target for neutralizing antibodies6,7,8. GP1,2 is expressed from the fourth of the seven filoviral genes, GP. In the case of marburgviruses, GP1,2 is the only GP expression product. In the case of cuevaviruses and ebolaviruses, the primary GP expression product is a non-structural secreted glycoprotein of unknown function (sGP). GP1,2 and another non-structural secreted glycoprotein (ssGP) are expressed via co-transcriptional mRNA editing resulting in addition of one or two adenosyls into the mRNA, respectively, thereby leading to open reading frame switches9,10,11,12 (Fig. 1). Filoviral GP1,2 is expressed akin to typical preproproteins. The primary expression product is steered into the endoplasmic reticulum (ER) by its signal peptide. Signalase cleaves off the signal peptide to yield preGP, and a host protease, furin, cleaves preGP into two subunits, GP1 and GP2, that remain linked by disulfide bonds (GP1,2)8,13,14.
Despite the virulence of filoviruses, most filoviruses are not thoroughly characterized, and comparatively few commercially produced reagents are available for their study15,16. For instance, availability of soluble filovirus GP1,2 is necessary for a variety of applications, including greater understanding of GP1,2-receptor or GP1,2-antibody binding kinetics, vaccine development, and GP1,2 structural studies. An ectodomain of the EBOV (variant Yambuku, isolate Mayinga) GP1,2 has been used as part of commercially available ELISAs for quantitation of antibody responses17, and a soluble, modified EBOV Yambuku-Mayinga GP1,2 ectodomain has been used to determine the crystal structure of GP1,2 bound to NPC118. The ectodomain of both EBOV8 and MARV19 with mucin-like domain deletions have been produced previously for crystallization studies. Additionally, efforts such as vaccine development use different organismal cell types as platforms to produce filovirus GP1,2, including mammalian cells20,21,22 and insect cells23. Specifically, authors have successfully used the Sf9-baculovirus system to produce full-length Ebola glycoproteins for use in VLPs23 and nanoparticle vaccines24,25. Other groups have also successfully used poly-histidine (6xHis) tags to purify full-length EBOV glycoproteins26. Some insect-derived filovirus (predominantly EBOV) GP1,2s are commercially available, but most mammalian-derived filovirus GP1,2s are not.
To close gaps in filovirus GP1,2 availability, we report on two systems for production and purification of filovirus GP1,2s in insect (Sf9) and mammalian (human) cell lines, respectively. Using these systems, we have successfully expressed EBOV, BDBV, TAFV, SUDV, MARV, and LLOV GP1,2s and developed techniques for rapid production of soluble variants thereof. We recently used these techniques to successfully produce ebolavirus GP1,2s for glycosylation analysis of their glycans27. Our expression systems may be broadly applicable for production and affinity purification of other soluble proteins from insect and mammalian cells.
We also demonstrate here that the ebolavirus GP1,2 proteins obtained using the two systems have important differences in glycosylation. These differences encompass the number of glycans, the type of glycan species, and the distribution of glycans at specific sites. We consider that these data to have implications for downstream usages of the produced proteins, such as binding assays, where glycosylation of the proteins may impact function.
Modification of filovirus GP1,2 sequences
We made changes to the sequence of the filovirus glycoproteins of interest to aid in expression and purification, including: mutating the furin cleavage site for purification of GP1,2 complexes; truncating the GP2s upstream of the transmembrane domain to produce soluble GP1,2 complexes; mutating the editing site of the GP genes to ensure exclusive expression of GP1,2 complexes; and adding 6xHis tags to the C-termini of GP2s for purification GP1,2 complexes (Fig. 2).
Generation of baculoviruses containing modified filovirus GP1,2s
GP genes were synthesized with the modifications discussed above and cloned into pFastBac1 plasmids to generate bacmids for transfection. After transfection of bacmids into the Sf9 cells, expressed GP1,2s produced were verified by western blot and plaque purified to ensure that clonal baculovirus was used for infections.
Purification of Sf9-produced GP1,2s using nickel columns
As the GP1,2s produced by the baculovirus-infected Sf9 cells do not contain a transmembrane region, all GP1,2s were released into Sf9 culture media. Total media were collected at day 3 post-transfection, replaced, collected again at day 4, and pooled together for purification. For each purification, between 2–4 T150 flasks of Sf9 cells were used for overall final yields ranging from 400 µg to 800 µg of GP1,2.
Purified proteins were verified by western blot using antibodies against either the specific GP1,2, or against the His-tag in the case of LLOV GP1,2. Purity was confirmed by periodic acid-Schiff (PAS) staining (preservation of glycosylation patterns) and by Colloidal Blue staining for detection of overall protein. Using this method of production, high levels of GP1,2 purity were obtained with the nickel column (Fig. 3b,c, Supplementary Fig. S1).
Additionally, we attempted protein production in a different insect cell line, High Five cells (ATCC CRL-10859), which have been reported28 to produce greater amounts of protein upon infection with baculoviruses. In our small-scale experiments, High Five cells did not yield greater protein than Sf9 cells, but this yield is likely to protein specific (data not shown).
Expression of filovirus GP1,2 in HEK 293T cells
Modified GP genes were cloned into pcDNA3.1+ backbones using the CMV enhancer-promoter to drive high levels of mammalian expression. BDBV, EBOV/Makona, EBOV/Yambuku, SUDV, TAFV, and LLOV GP1,2s were all successfully produced 3 days after Jet Prime transfection of the pcDNA3.1+ vector.
Plasmid vector backbone affects expression level of proteins
In our experiments, MARV/Musoke GP1,2 could not be expressed using pcDNA3.1+. The sequence of the MARV/Musoke GP gene used was truncated at residues encoding amino acid 636 (preGP numbering), i.e., upstream of the predicted transmembrane region29.This sequence was identical to the sequence used in the MARV/Musoke GP gene cloned into the baculovirus that successfully produced MARV GP1,2 in the insect cell system. Likewise, expression of the MARV/Musoke GP gene encoding a GP1,2 truncated to encode only amino acid residues 1–648 or 1–644 was unsuccessful in the pcDNA3.1+ background (Table 1). In contrast to commercially available MARV GP1,2 expression plasmids, which use codon-optimized GP genes for expression in mammalian cells, all our modified sequences were based on the wild-type GP sequences. Transfer of the MARV/Musoke GP gene sequence from pcDNA3.1+ into the pCAGGs expression vector did not lead to successful expression of MARV GP1,2, despite successful expression of unmodified full-length MARV GP by pCAGGs and not pcDNA3.1+ (Table 1, Supplementary Fig. S2). However, MARV/Angola (1–648) GP1,2 could be successfully expressed from pCAGGs (Table 1, Supplementary Fig. S2).
Mammalian HEK 293T culture conditions are incompatible with nickel column purification
After verifying successful filovirus GP1,2 expression from plasmids, we transfected plasmids into HEK 293T cells for purification and harvested media on day 3 post-transfection. Initially, we again used the HisTrap Excel Nickel Column for purification. However, elutions from the column contained an additional contaminating band (Fig. 4b) of approximately 80 kDa. This protein was glycosylated, as determined by PAS staining, but did not react with antibodies against GP1,2.
We attempted to remove the contaminant through multiple methods. Initial attempts to modify the HisTrap Excel protocol by doubling the wash buffer volume, increasing imidazole concentration in wash buffer, and increasing imidazole concentration in sample did not eliminate the contaminant. We used 8 M of urea to dissociate a possible protein-protein interaction with the contaminant. However, running the supernatant on a native gel revealed that the contaminating protein was not interacting with the GP1,2s. Due to the size difference between the GP1,2s and the contaminant, we also attempted dialysis of the elution with membranes with pore sizes of 100 kDa, 300 kDa, or 1,000 kDa. We then used size exclusion columns to remove the contaminant, but without success. We tried altering the purification method first by using a His-streptavidin tag-enrichment kit with a streptavidin column and then by replacing the nickel column with a cobalt column. Both approaches have been reported to reduce binding of contaminating proteins due to lower affinity for the His-tag. Yet, these approaches were not effective. The contaminating protein was identified by mass spectrometry as bovine serotransferase, a component of the fetal bovine serum (FBS) used in the HEK 293T cell media.
Purification of HEK 293T cell-produced GP1,2s with anti-His affinity resin columns
Anti-His Affinity Resin (GenScript) was used for purification of filovirus GP1,2 to avoid bovine serotransferase protein contamination. T150 flasks containing HEK 293T cells at 50–80% confluency were transfected with plasmids and, on day 3 post-transfection, media were collected for use in purification. Each purification on the Anti-His Affinity Resin gave an overall protein yield ranging from 150 µg to 500 µg, depending on GP1,2. Different GP genes resulted in different GP1,2 expression levels, with EBOV/Makona and EBOV/Yambuku genes expressing more GP1,2s than those of other filoviruses in both insect and mammalian cells (data not shown). Purified GP1,2s were again verified by western blot, and purity was confirmed by PAS and Colloidal Blue staining (Fig. 4c–e). Using this approach for glycan analysis, we confirmed the uniformity of the individual GP1,2s produced, demonstrating consistent GP1,2 glycosylation over multiple batches27.
Enzymatic digest of glycans from HEK 293T and SF9-cell produced GP1,2
EBOV/Makona GP1,2 produced in HEK 293T cells has a molecular weight by electrophoresis of around 160kDa, compared to just ≈110 kDa molecular weight of Sf9-produced EBOV/Makona (Fig. 5a,b). To demonstrate the contribution to molecular weight from glycosylation, we removed N-linked glycans from both proteins using PNGase F, and showed that ≈31% of 160 kDa HEK 293T cell-derived EBOV/Makona GP1,2, or 50 kDa total molecular weight, are N-linked glycans, compared to ≈35%, or ≈38 kDa of 110 kDa Sf9 cell-derived EBOV/Makona GP1,2. We then used a deglycosylation mix of enzymes that removes the majority of N and O-linked glycans and found that an additional ≈24%, or 38 kDa, of the HEK 293T cell-derived protein are O-linked glycans. However, the deglycosylation enzyme mix did not further reduce the molecular weight of the Sf9-derived EBOV/Makona protein, suggesting little or no observable O-linked glycosylation on the insect cell derived-proteins.
Analysis of N-linked glycans in HEK 293T and Sf9 cell produced GP1,2s
Our group has previously published in detail on the composition of the N-glycans on ebolavirus GP1,2 27. The broad differences in ebolavirus GP1,2 produced in HEK 293T cells are summarized in Fig. 5c. Briefly, the majority of the N-linked glycans are of the complex type, with few high mannose and hybrid glycans. The majority of the glycan species imparted on the mammalian GP1,2s are fucosylated. Di-antennary N glycans which are the dominant complex glycan structures for both EBOV GP1,2 samples are significantly less represented for BDBV, TAFV and SUDV GP1,2. The latter proteins showed increased levels of tri- tetra-, and penta- antennary N-glycans. Sialylated N-glycans correspond in all HEK 293T derived GP1,2 samples to about 15% of total N-glycans. Analysis of the Sf9-cell derived ebolavirus GP1,2 proteins reveals simpler glycan profiles, with a fucosylated core N-glycan (Fuc)1 (Man)3 –GlcNAc)2 as the largely dominant structure and some high-mannose type glycans (Fig. 5d). The N-glycans found on Sf9 cell derived GP 1,2 were in accordance with the glycosylation potential of this expression system30 lacking notably complex galactosylated or sialylated glycans.
Analysis of O-linked glycans in HEK293T and Sf9 cell-produced GP1,2s
The GP1,2s of filoviruses are predicted to have extensive O-linked glycosylation in addition to N-linked glycosylation. We have previously shown that ebolavirus GP1,2s produced in HEK 293T cells are extensively glycosylated with O-linked glycans, although the individual O-glycans imparted vary among different ebolaviruses, including between the two strains of EBOV, Makona and Yambuku27. To investigate the differences between GP1,2s produced in insect and mammalian cell types, we analyzed O-linked glycosylation of Sf9-derived ebolavirus GP1,2s. Primary analysis of ebolavirus GP1,2s generated in insect cells by mass spectrometry analysis of released permethylated O-glycans did not reveal any classical O-glycans for EBOV/Makona, EBOV/Yambuku, BDBV, TAFV and SUDV GP1,2s. However, monosaccharide quantification of EBOV Makona GP1,2 from Sf9 cells was performed to verify the presence of N-acetylgalactosamine (GalNAc) residues. These residues might be indicative of the presence of simple O-glycan structures on the protein that had not been detectable by O-glycan profiling of permethylated glycans. The observed presence of a low amount of GalNAc (measured as Galactosamine, GalN) of 3.3 nmol per nmol of protein allows us to presume that EBOV Makona from Sf9 cells carries a simple O-glycosylation essentially consisting of GalNAc residues. The presence of 1nmol of galactose per nmol of protein may indicate that some (Gal)1 (GalNAc)1 structures are present. These data suggest that insect-derived filovirus GP1,2 carry minimal levels of O-glycans that consist primarily of single GalNAc residues, in comparison to the extensively O-glycosylated HEK 293T-derived ebolavirus GP1,2s.
N-glycan site occupancy for EBOV/Makona GP1,2 produced in HEK 293T cells and Sf9 cells
The GP1,2 protein of EBOV/Makona contains 17 potential N-glycosylation sites that are represented in Fig. 6, obtained by analysis of the sequence using database search on EXPAZY Glycomod. The prediction of glycosylation sites does not differentiate based on cell or organism type but is only based on the presence of the N-glycosylation sequon Asn -X-Ser/Thr within the protein sequence. In a preliminary study combining the analysis of glycopeptides prior to and after enzymatic glycosylation, occupation of most of the 17 theoretical N-glycosylation sites with N-glycans could be observed. Sf9-derived EBOV/Makona GP1,2 contained15/17 detectable occupied sites while HEK 293T-derived EBOV/Makona GP1,2, revealed the presence of 13/17 detectable occupied sites. At the present level of analysis we cannot exclude that the remaining theoretical glycosylation sites are also occupied since the relatively large sizes of the corresponding tryptic glycopeptides may have prohibited their analysis by MALDI-TOF mass spectrometry. There are significant differences in the variety of different glycan species found at any given N-glycan occupied site, with the HEK 293T-derived EBOV/Makona GP1,2 having a greater number, up to 13 different N-glycans at an individual site (N172 corresponding to amino acid 204 of the construct), compared to the Sf9-derived, which had a maximum of 4 N-glycans at a given site (N264 corresponding to amino acid 296 of the construct). The heterogeneity of site specific N-glycosylation between mammalian and insect cell derived GP1,2 is in accordance with the observed differences of overall N-glycan profiles of proteins produced in these expression systems. Significantly, the two predicted glycosylation sites in GP1,2 shown to be necessary for VSV pseudotype entry and production31 of full-length EBOV/Mayinga GP1,2, were also found to be glycosylated in our EBOV/Makona GP1,2 produced by either cell type.
In the present study, we demonstrate two techniques for producing and purifying filovirus GP1,2s. We modified the GP gene sequences from ebolaviruses, MARV, and LLOV to express released GP1,2s by truncating the sequences to remove the transmembrane domains. For higher expression, we altered the RNA-editing sites of the ebolavirus and LLOV GP genes (Fig. 2). This approach prevented expression of sGP and ssGP, which could interfere with growth of cells in culture and downregulate GP1,2 expression32. EBOV GP1,2 has been reported to be cytotoxic when expressed at high levels33,34, which would not be ideal for protein production. However, this property has been attributed to the transmembrane region of the protein35, and thus deleting the transmembrane domain may have circumvented this potential problem.
We mutated the furin cleavage sites of filovirus GP1,2s to prevent dissociation of the GP1 and GP2 subunits during the purification process (Fig. 2). This step enabled maintenance of equal expression levels of GP1 and GP2 and circumvented purification of only GP2 (which contained the His-tag). However, we did not codon-optimize the GP gene sequences because codon optimization is known to increase protein expression levels, thereby possibly leading to cytotoxicity33,34. The modified sequences were cloned into either pcDNA3.1+, a commonly used expression plasmid for mammalian cell transfections, or into a shuttle vector, pFastBac1, for baculovirus (AcMNPV) production. Insect cells are a commonly used system for protein production in part because of the success of baculovirus systems. Baculoviruses are versatile vectors for protein production in insect cells that are easily scaled up for high output28,36.
We successfully expressed modified ebolavirus and LLOV GP1,2s in HEK 293T cells using the pcDNA3.1+ expression plasmid. However, we were unable to successfully produce MARV GP1,2 in the HEK 293T cell system using pcDNA3.1+ (Table 1). Non-modified full-length MARV GP1,2 was also not successfully produced in the HEK 293T system using the pcDNA3.1+ vector but was produced using the vector pCAGGs despite the encoded sequences being identical. Further, using the pCAGGs expression vector, we could not express modified MARV/Musoke GP1,2, but could express modified MARV/Angola GP1,2 (Table 1, Supplemental Fig. 2). The amino acid sequence difference between the two glycoproteins is 7.2%37. Thus, the vector choice is gene-specific in the mammalian system, even among genes from related viruses. Therefore, attempting expression with multiple vectors may be necessary for a protein of interest.
Attempts to purify mammalian-derived GP1,2 through the HisTrap Excel Nickel Column system that we used for Sf9 purification were unsuccessful due to a component of the FBS that binds to the nickel column (Fig. 4b). This component was identified as bovine serotransferase. To our knowledge, this paper is the first report of an incompatibility of the nickel column system and mammalian tissue culture that uses FBS. The Anti-His Affinity Resin approach that we detail here may therefore be applicable to purification of many mammalian cell-produced proteins. Another alternative may be the use of serum-free 293T cell culture media. In summary, we demonstrated that different purification techniques are required for mammalian and insect filovirus GP1,2 production.
We demonstrate that there are key differences in the type of glycosylation imparted on the ebolavirus GP1,2s produced using the two methods outlined, for insect and mammalian cell culture. Key differences in complexity of the N-linked glycans between the two systems were found for all of the ebolavirus GP1,2s. The mammalian derived-proteins demonstrated high percentages of complex N-linked glycans compared to the majority of simple N-linked glycans and high mannose structures typically found in SF9 insect cell derived protein. The HEK 293T derived GP1,2s showed some heterogeneity in the complexity of antennary structures with the two EBOV samples containing predominantly di-antennary complex glycans and BDBV, TAFV and SUDV harboring complex glycans with higher antennary structures. The mammalian system also produced N-glycans that were sialylated. Although it remains controversial whether some insect protein production systems are able to impart sialylation, with some well controlled studies suggesting that they can at a minimal level38. Using our Sf9 cell production system, we did not see sialylation on any insect-derived N-linked glycans. As sialylation has been shown to play an important role in the function of some glycoproteins, it is possible that this difference could impart significant functional differences39.
Insect cell ability to impart O-linked glycans has been controversial, with some studies demonstrating limited O-linked glycans present on proteins derived from Sf9 cells40. Our analysis reveals the possibility of limited GalNAc-only O-linked glycans on the EBOV/Makona analyzed by monosaccharide quantification. However, while there is some evidence of limited O-linked glycosylation from insect cell-produced GP1,2s, mammalian cell O-linked glycosylation is extensive, a key difference between the two systems of production. For lesser studied ebolaviruses such as Bundibugyo and Taї Forest viruses, few reagents are available, and the few commercially available proteins are often produced in insect cells. We demonstrate that the post-translational modifications are different in mammalian compared to insect systems for production of filovirus GP1,2s. A comparison between the two systems may be useful in deciding how to produce large-scale amounts of protein for purposes such as vaccination, enzyme-linked immunosorbent assays, or the study of receptor or antibody binding kinetics. Our approaches successfully produced filovirus GP1,2s in both systems, therefore allowing phenotypic comparisons between the expressed proteins.
Materials and Methods
Cells and plasmid vectors
Fall armyworm (Spodoptera frugiperda) Sf9 cells (Invitrogen, Germany) were grown at 27 °C in Sf-900II serum-free medium (Gibco, USA) with 100 U/ml of penicillin and 100 µg/ml of streptomycin in the suspension. Human embryonic kidney (HEK) 293T cells (ATCC CRL-3216, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS) and 1% penicillin-streptomycin.
We created GP1,2-expressing plasmids based on GP genes from one cuevavirus, four ebolaviruses, and two marburgviruses. All but two plasmids are based on reference GP sequences: Lloviu virus/M.schreibersii-wt/ESP/2003/Asturias-Bat86 (“LLOV”; RefSeq #NC_016144), Bundibugyo virus/H.sapiens-tc/UGA/2007/Butalya-811250 (“BDBV”; RefSeq #NC_014373), Ebola virus/H.sapiens-tc/COD/1976/Yambuku-Mayinga (“EBOV/Yambuku”; RefSeq #NC_002549), EBOV/H.sapiens-wt/GIN/2014/Makona-C07 (“EBOV/Makona”, GenBank #KJ660347), Sudan virus/H.sapiens-tc/UGA/2000/Gulu-808892 (“SUDV”; RefSeq #NC_006432), Taï Forest virus/H.sapiens-tc/CIV/1994/Pauléoula-CI (“TAFV”; RefSeq #NC_014372), Marburg virus/H.sapiens-tc/KEN/1980/Mt. Elgon-Musoke (“MARV”; RefSeq #NC_001608), and Marburg virus/H.sapiens-tc/AGO/2005/Angola-20051379 (“MARV/Angola”, GenBank #KU978782). All genes were synthesized by Genscript (Piscataway, NJ) or cloned at Biosafety level 2 by the authors from inactivated filovirus cultures. All experiments were performed in Biosafety level 2 conditions.
Glycoprotein sequence modifications
A number of GP gene modifications was required to produce soluble filovirus GP1,2s. An additional adenosyl was added to the cuevavirus and ebolavirus 7A co-transcriptional editing sites (yielding 8A-GP) to ensure expression of GP1,2 in the absence of sGP and ssGP production41. The coding regions for the GP2 transmembrane regions were removed to ensure secretion. Truncation occurred at amino acid 692 (LLOV preGP numbering), 650 (ebolavirus preGP numbering), and 638 (MARV preGP numbering). A region encoding a C-terminal polyhistidine (6xHis) tag was added to all genes to aid purification of expressed proteins through affinity chromatography. Finally, furin cleavage of preGPs was prevented by site-directed mutagenesis leading to a single arginine-to-lysine change in the VYFRRKR furin cleavage site. This change is known not to affect the protein’s function42, which is why we continue to refer to GP1,2s in this manuscript.
Modified GP genes were cloned into the pcDNA3.1+ mammalian expression vector and transfected into HEK 293T cells at 50–80% confluency using JetPRIME transfection reagent (Polyplus transfection, New York, NY). Modified GP genes were also cloned into pFastBac™1 (ThermoFisher Scientific, Waltham, MA), a transposing vector with the baculovirus Autographa californica multicapside nucleopolyhedrovirus (AcMNPV) polyhedrin (PH) promoter. The pFastBac™1 constructs were then transformed in MAX Efficiency® DH10Bac™-competent cells (ThermoFisher Scientific, Waltham, MA), generating recombinant bacmids for transfection into Sf9 cells using Cellfectin® II Reagent (ThermoFisher Scientific, Waltham, MA). AcMNPVs expressing GP1,2s were verified for correct orientation 72 h after transfection of Sf9 cells in 6-well plates by western blot of cell supernatants. Supernatants containing AcMNPVs was then used for plaque purification with 10-fold dilutions. Individual plaques were purified by adding an agarose plug to a T150 Sf9 flask and incubating for 4 days. Supernatants from these flasks were titered using plaque assays of 95% confluent Sf9 cells and a 4% agarose overlay.
Purification of Sf9-produced glycoproteins
Sf9 cells were infected with AcMNPVs at a multiplicity of infection of 0.01. Media were harvested on days 3 and 4 post-inoculation and pooled. Modified GP1,2s were purified using the HisTrap excel system (GE Healthcare Bio-Sciences, Pittsburgh, PA), following the manufacturer’s instructions. Briefly, Sf9 supernatants were passed through a Ni2-chelated HisTrap Excel column (GE Healthcare) using a variable flow mini-pump (Fisher Scientific). The column was washed with wash buffer (20 mM of sodium phosphate, 0.5 M of NaCl and 20 mM of imidazole [GE Healthcare]) and eluted in 5 ml of elution buffer (20 mM of sodium phosphate, 0.5 M of NaCl, and 500 mM of imidazole).
Purification of glycoproteins from HEK 293T cells
Anti-His Affinity Resin (GenScript) was used for purification of GP1,2s following the manufacturer’s instruction. Briefly, resin was incubated with supernatant and rotated for 30 min and resins were placed into Econo-Pac Disposable Chromatography columns (BioRad, Hercules, CA) and washed with Tris-buffered saline (pH 7.4). Resins were then incubated with hexa-His peptide (GenScript) at a concentration of 0.5 µg/ml. Eluents containing proteins of interest were concentrated using Pierce Protein Concentrator (Thermo Fisher Scientific, Waltham, MA) for downstream use.
Verification of protein purity
Protein purity was verified via a Colloidal Blue stain, PAS, (ThermoFisher Scientific, Waltham, MA), and western blot. Purified proteins were run on 4–12% Bis-Tris Plus polyacrylamide gels (Life Technologies, Carlsbad, CA). Gels were then stained using PAS to detect GP1,2s according to the manufacturer’s instructions. Total protein was then assayed using the Colloidal Blue staining kit (ThermoFisher Scientific, Waltham, MA). Finally, western blots were performed by transferring gels, as above, to iBlot 2 nitrocellulose Mini Stacks (Life Technologies) using the iBlot2 system (Life Technologies).
Membranes were blocked using 5% dry milk in phosphate buffered saline and 0.01% Tween 20 (PBST, Sigma-Aldrich) for 1 h at room temperature. Membranes were then incubated with primary antibody in 5% milk in PBST for 1 h with shaking. Antibody dilutions were as follows: BDBV (rabbit polyclonal, IBT Bioservices, Rockville, MD, USA): 1:5,000; EBOV (6D8, courtesy of John Dye, US Army Medical Research Institute of Infectious Diseases, Frederick, MD): 1:10,000; TAFV (rabbit polyclonal, Alpha Diagnostic International, San Antonio, TX): 1:1,000; SUDV (clone 6D11, BEI Resources, Manassas, VA): 1:5,000; MARV (rabbit polyclonal, IBT Bioservices, Rockville, MD): 1:3,000; and THETM His Tag Antibody (mouse monoclonal, Genscript). Blots were then washed thrice with PBST and incubated and shook for 1 h at room temperature with secondary antibody. Secondary antibodies were anti-mouse IgG-horseradish peroxidase (HRP) or anti-rabbit IgG-HRP (ThermoFisher Scientific, Waltham, MA), both at 1:5,000 dilution. Blots were then washed three times with PBST, and a 1:1 mixture of Novex ECL HRP Chemiluminescent Substrate (ThermoFisher Scientific, Waltham, MA) was added to the membranes for development using a medical film processor (Konica Minolta Medical Imaging, Wayne, NJ).
Enzymatic removal of glycans
Glycans were removed from purified GP1,2s using PNGase F (New England BioLabs, Beverly, MA) and deglycosylation mix (New England BioLabs, Beverly, MA) according to manufacturer’s instructions. Proteins were imaged using Colloidal Blue analysis, as above, and approximate percentages calculated using Novex Sharp Prestained Protein ladder (ThermoFisher Scientific, Waltham, MA).
N-glycan release by PNGase F for glycan analysis
Glycan composition analysis was performed by Proteodynamics (Riom, France). N-linked glycans were removed from HEK 293T derived GP1,2 using 15 U per 200 µg protein of PNGase F (Promega, Madison, WI, USA) for 15 hours at 37 °C in phosphate buffer (pH of 7.5) while Sf9 cell derived protein was deglycosylated with 15 U of PNGase F (Promega, Madison, WI, USA) and 10 µL of PNGAse A (Sigma) for 15 hours at 37 °C in 50 mM sodium acetate buffer, pH 5.0. To confirm deglycosylation, electrophoresis on NuPAGE 4–12% gels (ThermoFisher Scientific, Waltham, MA) was performed. The N-glycans were then purified and permethylated according to Morelle et al.43.
O-glycan release by beta-elimination for glycan analysis
Glycoproteins (200 µg) were neutralized and loaded onto beads and then lyophilized overnight, and then suspended in 200 µL of MilliQ water and 200uL reductive solution [100 mM NaOH, 2 M NaBH4]. Beta-elimination occurred for 15 hours at 45 °C to release O-glycans. Permethylation was performed as for the N-glycans43.
Glycan composition analysis
Once permethylated, purified glycans were solubilized in 20 µL of a 1:1 ratio methanol/DI water mix. A mix of 2 µL non-dilute and 2 µL N-glycans with 2 µL of 2,5-dihydroxybenzoic acid (LaserBio Labs, Sophia-Antipolis, France) matrix solution (10 mg/ml in 1:1 ratio methanol/DI water). Positive ion reflectron MALDI-TOF mass spectra were acquired using an Autoflex III mass spectrometer (Bruker Daltonics, Billerica, MA, USA). The acceleration and reflector voltage conditions were voltage 12 × 1977 V and 90% laser, and the spectra obtained by accumulation of 2,000 shots. The spectra were calibrated with an external standard (PepMix 4, LaserBioLabs, Sophia-Antipolis, France). To elucidate the glycan profiles of the sample, several spectra were obtained from different spots and the values averaged. To interpret the structures of the glycans corresponding to monisotopic masses after deisotoping of the spectra, the EXPAZY GlycoMod tool was used, along with GlycoWorkBench. Relative intensities of glycans were calculated to establish the glycan profile for each spectrum and mean values for the glycan intensities with standard deviations were determined.
N-glycan site occupancy analysis on EBOV-Makona from Sf9-cells and HEK 293T cells
Glycopeptide enrichment method
EBOV/Makona GP1,2 proteins from Sf9 and HEK293T (200 µg each) were denatured, reduced by DTT, and alkylated by iodoacetamide and digested by trypsin/Lys-C Mix (Promega, Madison, WI, USA) during 15 hours at 37 °C.according to Rapigest® SF protocol. Glycopeptides were enriched according to ProteoExtract® Glycopeptide Enrichment Kit protocol 72103-3 (Novagen, Madison, WI, USA). 30 µl of glycopeptide sample were added to 150 µl of ZIC Glycocapture Resin (Merck SeQuant AB, Umea, Sweden), and eluted with 225 µl ZIC Elution Buffer (Merck SeQuant AB, Umea, Sweden). The eluted samples were completely evaporated in with a SpeedVac concentrator (ThermoFisher Scientific, Waltham, MA).
MALDI-TOF MS analysis of site occupancy
Enriched glycopeptides (15 µl) in 50% methanol were mixed (1:1) with 2,5-dihydroxybenzoic acid (LaserBio Labs, Sophia-Antipolis, FR) matrix solution (10 mg/ml in 1:1 ratio methanol/water). Positive ion linear MALDI mass spectra were acquired on MALDI-TOF/TOF Autoflex Speed (Bruker Daltonics, Billerica, MA, USA). Acquisition conditions used, 10 × 2850 V, laser 25% and 2000 shots.
Deglycosylation of the enriched fraction
To remove glycans from the peptides, 5 µl of suspended glycopeptides were adjusted to 10 mM sodium acetate buffer pH 5 and 1 µl of PNGase A and 0.7 µl of PNGase F (Promega, Madison, WI, USA) or PNGase A (for insect cell protein) were added to deglycosylate during 15 hours at 37 °C. The deglycosylated peptides after preparation on Zip Tips C18 (EMD Millipore, Billerica, MA, USA) were mixed (1:1) with CHCA (LaserBio Labs, Sophia-Antipolis, France) matrix solution (7 mg/ml 50:50 acetonitrile/water, 0.1% TFA). Positive ion reflectron MALDI mass spectra were acquired on MALDI-TOF/TOF Autoflex Speed (Bruker Daltonics, Billerica, MA, USA). Acquisition conditions were 10 × 1950 V, laser 30% with 2000 shots.
Bukreyev, A. A. et al. Discussions and decisions of the 2012–2014 International Committee on Taxonomy of Viruses (ICTV) Filoviridae Study Group, January 2012–June 2013. Archives of virology 159, 821–830, https://doi.org/10.1007/s00705-013-1846-9 (2014).
Kuhn, J. H. In Harrison’s Principles of Internal Medicine (eds Dennis L. Kasper et al.) Ch. 234, 1323–1329 (McGraw-Hill Education, 2015).
Negredo, A. et al. Discovery of an ebolavirus-like filovirus in europe. PLoS pathogens 7, e1002304, https://doi.org/10.1371/journal.ppat.1002304 (2011).
Cote, M. et al. Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola virus infection. Nature 477, 344–348, https://doi.org/10.1038/nature10380 (2011).
Carette, J. E. et al. Ebola virus entry requires the cholesterol transporter Niemann-Pick C1. Nature 477, 340–343, https://doi.org/10.1038/nature10348 (2011).
Corti, D. et al. Protective monotherapy against lethal Ebola virus infection by a potently neutralizing antibody. Science 351, 1339–1342, https://doi.org/10.1126/science.aad5224 (2016).
Howell, K. A. et al. Antibody treatment of Ebola and Sudan virus infection via a uniquely exposed epitope within the glycoprotein receptor-binding site. Cell Rep 15, 1514–1526, https://doi.org/10.1016/j.celrep.2016.04.026 (2016).
Lee, J. E. et al. Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor. Nature 454, 177–182, https://doi.org/10.1038/nature07082 (2008).
Maruyama, J. et al. Characterization of the envelope glycoprotein of a novel filovirus, lloviu virus. Journal of virology 88, 99–109, https://doi.org/10.1128/JVI.02265-13 (2014).
Mehedi, M. et al. A new Ebola virus nonstructural glycoprotein expressed through RNA editing. Journal of virology 85, 5406–5414, https://doi.org/10.1128/JVI.02190-10 (2011).
Sanchez, A., Trappier, S. G., Mahy, B. W. J., Peters, C. J. & Nichol, S. T. The virion glycoproteins of Ebola viruses are encoded in two reading frames and are expressed through transcriptional editing. Proc Natl Acad Sci USA 93, 3602–3607 (1996).
Volchkov, V. E. et al. GP mRNA ofEbola virus is edited by the Ebola virus polymerase and by T7 and vaccinia virus polymerases. Virology 214, 421–430 (1995).
Volchkov, V. E., Feldmann, H., Volchkova, V. A. & Klenk, H.-D. Processing of the Ebola virus glycoprotein by the proprotein convertase furin. Proc Natl Acad Sci USA 95, 5762–5767 (1998).
Volchkov, V. E. et al. Proteolytic processing of Marburg virus glycoprotein. Virology 268, 1–6, https://doi.org/10.1006/viro.1999.0110 (2000).
Burk, R. et al. Neglected filoviruses. FEMS microbiology reviews 40, 494–519, https://doi.org/10.1093/femsre/fuw010 (2016).
Anthony, S. M. & Bradfute, S. B. Filoviruses: One of These Things is (not) Like the Other. Viruses 7, 5172–5190, https://doi.org/10.3390/v7102867 (2015).
Vu, H. et al. Quantitative serology assays for determination of antibody responses to Ebola virus glycoprotein and matrix protein in nonhuman primates and humans. Antiviral Res 126, 55–61, https://doi.org/10.1016/j.antiviral.2015.11.012 (2016).
Wang, H. et al. Ebola viral glycoprotein bound to its endosomal receptor Niemann-Pick C1. Cell 164, 258–268, https://doi.org/10.1016/j.cell.2015.12.044 (2016).
Hashiguchi, T. et al. Structural basis for Marburg virus neutralization by a cross-reactive human antibody. Cell 160, 904–912 (2015).
Stanley, D. A. et al. Chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge. Nature medicine 20, 1126–1129, https://doi.org/10.1038/nm.3702 (2014).
Falzarano, D. et al. Single immunization with a monovalent vesicular stomatitis virus-based vaccine protects nonhuman primates against heterologous challenge with Bundibugyo ebolavirus. The Journal of infectious diseases 204(suppl. 3), S1082–1089, https://doi.org/10.1093/infdis/jir350 (2011).
Daddario-DiCaprio, K. M. et al. Cross-protection against Marburg virus strains by using a live, attenuated recombinant vaccine. Journal of virology 80, 9659–9666, https://doi.org/10.1128/JVI.00959-06 (2006).
Warfield, K. L. et al. Filovirus-like particles produced in insect cells: immunogenicity and protection in rodents. The Journal of infectious diseases 196(Suppl 2), S421–429, https://doi.org/10.1086/520612 (2007).
Hahn, T. e. a. Rapid Manufacture and Release of a GMP Batch of Zaire Ebolavirus Glycoprotein Vaccine Made Using Recombinant Baculovirus-Sf9 Insect Cell Culture Technology. BioProcess Journal 14, 6–14, https://doi.org/10.12665/J141.Hahn (2015).
Bengtsson, K. L. et al. Matrix-M adjuvant enhances antibody, cellular and protective immune responses of a Zaire Ebola/Makona virus glycoprotein (GP) nanoparticle vaccine in mice. Vaccine 34, 1927–1935, https://doi.org/10.1016/j.vaccine.2016.02.033 (2016).
Melen, K. et al. Production, purification and immunogenicity of recombinant Ebola virus proteins− A comparison of Freund’s adjuvant and adjuvant system 03. Journal of virological methods 242, 35–45 (2017).
Collar, A. L. et al. Comparison of N- and O-linked glycosylation patterns of ebolavirus glycoproteins. Virology 502, 39–47, https://doi.org/10.1016/j.virol.2016.12.010 (2016).
Wilde, M., Klausberger, M., Palmberger, D., Ernst, W. & Grabherr, R. Tnao38, high five and Sf9—evaluation of host-virus interactions in three different insect cell lines: baculovirus production and recombinant protein expression. Biotechnol Lett 36, 743–749, https://doi.org/10.1007/s10529-013-1429-6 (2014).
Hevey, M., Negley, D., Geisbert, J., Jahrling, P. & Schmaljohn, A. Antigenicity and vaccine potential of Marburg virus glycoprotein expressed by baculovirus recombinants. Virology 239, 206-216, doi:S0042-6822(97)98883-8 [pii]10.1006/viro.1997.8883 (1997).
Shi, X. & Jarvis, D. L. Protein N-glycosylation in the baculovirus-insect cell system. Curr Drug Targets 8, 1116–1125 (2007).
Lennemann, N. J., Walkner, M., Berkebile, A. R., Patel, N. & Maury, W. The role of conserved n-linked glycans on ebola virus glycoprotein 2. The Journal of infectious diseases 212, S204–S209 (2015).
Gallaher, W. R. & Garry, R. F. Modeling of the Ebola virus delta peptide reveals a potential lytic sequence motif. Viruses 7, 285–305, https://doi.org/10.3390/v7010285 (2015).
Volchkova, V. A., Dolnik, O., Martinez, M. J., Reynard, O. & Volchkov, V. E. RNA editing of the GP gene of Ebola virus is an important pathogenicity factor. The Journal of infectious diseases 212(suppl. 2), S226–233, https://doi.org/10.1093/infdis/jiv309 (2015).
Volchkov, V. E. et al. Recovery of infectious Ebola virus from complementary DNA: RNA editing of the GP gene and viral cytotoxicity. Science 291, 1965–1969, https://doi.org/10.1126/science.1057269 (2001).
Hacke, M. et al. Inhibition of Ebola virus glycoprotein-mediated cytotoxicity by targeting its transmembrane domain and cholesterol. Nat Commun 6, 7688, https://doi.org/10.1038/ncomms8688 (2015).
Kost, T. A., Condreay, J. P. & Jarvis, D. L. Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23, 567–575, https://doi.org/10.1038/nbt1095 (2005).
Towner, J. S. et al. Marburgvirus genomics and association with a large hemorrhagic fever outbreak in Angola. Journal of virology 80, 6497–6516, https://doi.org/10.1128/JVI.00069-06 (2006).
Marchal, I., Jarvis, D. L., Cacan, R. & Verbert, A. Glycoproteins from insect cells: sialylated or not? Biological chemistry 382, 151–159 (2001).
Lehmann, F., Tiralongo, E. & Tiralongo, J. Sialic acid-specific lectins: occurrence, specificity and function. Cellular and molecular life sciences 63, 1331–1354 (2006).
Gaunitz, S. et al. Mucin-type proteins produced in the Trichoplusia ni and Spodoptera frugiperda insect cell lines carry novel O-glycans with phosphocholine and sulfate substitutions. Glycobiology 23, 778–796 (2013).
Mehedi, M. et al. Ebola virus RNA editing depends on the primary editing site sequence and an upstream secondary structure. PLoS Pathog 9, e1003677, https://doi.org/10.1371/journal.ppat.1003677 (2013).
Neumann, G. et al. Proteolytic processing of the Ebola virus glycoprotein is not critical for Ebola virus replication in nonhuman primates. Journal of virology 81, 2995–2998, https://doi.org/10.1128/JVI.02486-06 (2007).
Morelle, W. & Michalski, J. C. Analysis of protein glycosylation by mass spectrometry. Nature protocols 2, 1585–1602, https://doi.org/10.1038/nprot.2007.227 (2007).
The authors thank Laura Bollinger (NIH/NIAID Integrated Research Facility at Fort Detrick, Frederick, MD, USA) for critically editing the manuscript. This project was supported by Defense Threat Reduction Agency grant HDTRA1-15-1-0061 (SBB). The content of this publication does not necessarily reflect the views or policies of the US Department of the Army, the US Department of Defense, the US Department of Health and Human Services, or the institutions and companies affiliated with the authors. This work was funded in part through Battelle Memorial Institute’s prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272200700016I. An employee of Battelle Memorial Institute is: JW. Subcontractors to Battelle Memorial Institute who performed this work are: YC and JHK, employees of Tunnell Government Services, Inc.
The authors declare that they have no competing interests.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
About this article
Cite this article
Clarke, E.C., Collar, A.L., Ye, C. et al. Production and Purification of Filovirus Glycoproteins in Insect and Mammalian Cell Lines. Sci Rep 7, 15091 (2017). https://doi.org/10.1038/s41598-017-15416-3
npj Vaccines (2018)