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. Author manuscript; available in PMC: 2023 Feb 7.
Published in final edited form as: Methods Mol Biol. 2020;2060:377–393. doi: 10.1007/978-1-4939-9814-2_23

Expression, Purification, and Crystallization of HSV-1 Glycoproteins for Structure Determination

Ellen M White 1, Samuel D Stampfer 1, Ekaterina E Heldwein 1
PMCID: PMC9903252  NIHMSID: NIHMS1858285  PMID: 31617192

Abstract

Herpes simplex viruses utilize glycoproteins displayed on the viral envelope to perform a variety of functions in the viral infectious cycle. Structural and functional studies of these viral glycoproteins can benefit from biochemical, biophysical, and structural analysis of purified proteins. Here, we describe a general protocol for expression and purification of viral glycoproteins from insect cells based on those developed for the HSV-1 gB and HSV-2 gH/gL ectodomains as well as the protocol for crystallization of these glycoproteins. This protocol can be used for generating milligram amounts of wild-type (WT) or mutant gB and gH/gL ectodomains or can be adapted to produce purified ectodomains of glycoproteins from HSV or other herpesviruses for biochemical and structural studies.

Keywords: Herpes simplex viruses, Glycoproteins, Viral entry, Ectodomain, Crystallography, Protein purification

1. Introduction

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are enveloped viruses that display 12 glycoproteins and three nonglycosylated proteins on their surface. These proteins perform a variety of functions in the viral infectious cycle, including entry into the host cell by mediating membrane fusion, cell–cell spread, viral egress, and other yet unclear roles [1–3]. Being surface exposed, they also modulate the immune response, and some generate neutralizing antibodies [4, 5]. Their surface exposure and essential functions make them promising targets for the development of drugs and vaccines.

HSV glycoproteins gD, gH, gL, and gB are necessary [6–9] and sufficient [10] for viral entry into the host cell. The receptor-binding protein gD binds one of its three cellular receptors, which determines viral tropism [1, 11]. Binding to a cognate receptor triggers a conformational change in gD that is thought to lead to the activation of gH/gL and, ultimately, gB during the host cell fusion process [11, 12]. While the specific role of the heterodimer gH/gL in the membrane fusion process is unknown, it is thought to activate gB in response to an activating signal from gD [13, 14] and may function as a fusion adaptor protein [15]. gB is a class III viral fusogen [16] that undergoes a conformational rearrangement from the prefusion to the postfusion form [17, 18].

Most HSV glycoproteins are anchored to the membrane by at least one transmembrane region. The presence of a transmembrane anchor presents many challenges to isolation of glycoproteins. While detergents have successfully been used to solubilize integral membrane proteins, solubilization of membrane proteins containing large external portions has remained challenging. Thus, isolation of glycoprotein ectodomains has great appeal.

The ectodomains of several HSV glycoproteins, gD, gB, and the gH/gL heterodimer, have been successfully produced as purified proteins, which has been critical for understanding how they work. In particular, the ability to produce large quantities of homogenous proteins has allowed the determination of their crystal structures. The crystal structures of gD alone and bound to two different receptors have provided detailed knowledge of gD/receptor interactions [12, 19–22]. The crystal structures of gB and gH/gL, determined in our laboratory, have led us to propose that gB functions as a viral fusion protein [17] whereas gH/gL serves a fusion activator [23] during cell entry and cell–cell fusion, which was confirmed in subsequent studies. The structures of all four proteins have also aided the mapping of their functional regions [11]. Finally, the ability of purified gD and gH/gL ectodomains to function in cell–cell fusion assays, albeit with reduced efficiency, suggested that membrane anchoring is not essential for the function of these proteins during cell fusion [13, 24], consistent with their regulatory roles.

The ectodomains of gD, gH/gL, and gB were produced as secreted proteins in insect cells infected with recombinant baculoviruses. The use of baculovirus expression vector systems (BEVS) technology for expression of recombinant proteins, including glycoproteins, has been described elsewhere (e.g., [25]), and will not be covered here. The advantage of using insect cells is that unlike in bacterial cells, proteins expressed in insect cells can be properly folded, posttranslationally modified (glycosylated or proteolytically processed), and trafficked [25]. Insect cells are also relatively simple to propagate, can be easily grown in suspension cultures, and protein expression can be easily scaled up to large volumes at significantly lower cost than in mammalian cells. Finally, BEVS utilizes baculoviruses, which are easily manipulated and relatively safe to use. Using BEVS, recombinant genes of interest are expressed under the control of the polyhedrin promoter of baculovirus. To ensure that the glycoproteins are transported to the cell membrane or extracellular space after expression, their genes must include an endogenous or heterologous signal sequence, e.g., from honeybee melittin. Detailed instructions for recombinant baculovirus generation are listed in the Bac-to-Bac™ Baculovirus Expression System manual [26]. We typically expand the initial baculovirus stock in two passaging steps. The resulting passage 3, also referred to as P3, can be used to infect insect cells for protein production. While the protocol described here uses Sf9 insect cells, Sf21 or High Five™ insect cells can also be used.

Prior to expression, DNA fragments encoding the protein region of interest must be subcloned into a plasmid vector suitable for baculovirus generation. Although there are several BEVS systems currently on the market, we use the Bac-to-Bac™ Baculovirus Expression System (Thermo Fisher Scientific) [26] due to its simplicity and relative speed of generating recombinant baculoviruses because it does not require the use of a plaque assay. For the HSV-1 gB ectodomain, henceforth referred to as gB730, DNA encoding residues 31-730 was subcloned into pFastBac™ vector (Thermo Fisher Scientific), with the honeybee melittin signal sequence. Wild-type gB730 and several mutants and derivatives have been purified using this strategy [27–29], yielding 0.5–2 mg of purified protein per liter of insect cell culture.

The gH and gL glycoproteins must be co-expressed for proper complex formation [7, 23, 30]. Therefore, gH and gL were subcloned into the pFastBacDual™ vector (Thermo Fisher Scientific) that allows for expression of two genes from separate late baculovirus promoters. Two versions of the soluble gH/gL complex have been expressed using this approach: gH803-H6/gL, which contains the entire gH ectodomain, residues 19-803, with a C-terminal His6 tag and full-length gL, residues 1-224, [29]; and Δ48gH803-H6/gL, which lacks residues 19-47 of gH but is otherwise identical to gH803-H6/gL [23]. Δ48gH803-H6/gL is the only HSV gH/gL complex crystallized thus far [23] whereas both gH803-H6/gL and Δ48gH803-H6/gL have been used in functional assays [13, 31]. Each complex was produced using the same protocol that yields ~250 μg of homogeneous complex per liter of insect cell culture.

Here, we describe a general protocol for expression and purification of glycoproteins in Sf9 cells based on those developed for the ectodomains of HSV-1 gB730 (residues 31-730) and HSV-2 gH803-H6/gL (residues 19-803 of gH and full-length gL). gB is used as an example of a glycoprotein purified using immunoaffinity while gH/gL is an example of a glycoprotein purified through the use of an affinity tag. In this case, we used immobilized metal-ion affinity chromatography (IMAC) purification via a hexahistidine tag. Although yields of pure protein per liter of insect cells vary for the three glycoproteins, from 250 μg for gH/gL to 2 mg for gB to 10 mg for gD, all three have been produced in the amounts sufficient for crystallization and functional assays. Crystallization and freezing of crystals of HSV-1 WT gB730 and HSV-2 Δ48gH803-H6/gL (the construct described above, missing the first 47 residues of gH) is also described.

These protocols can be used for the production of the WT or mutant constructs of gB or gH/gL for biochemical, structural, or functional use. With slight modification, these protocols can be adapted for the production of other HSV glycoproteins or glycoproteins from other viruses.

2. Materials

2.1. Protein Expression

  1. A setup for growing insect cells in suspension in spinner flasks (see Note 1), including several 3-L spinner flasks with a 2-port airflow assemblies, a magnetic stir plate for spinner flasks, an air pump, a gas blending stand, ¼″ inner diameter autoclavable plastic tubing, and 0.2 μm air filter units (Millex-FG50 or similar). Available as a kit from Bellco or may be purchased separately.

  2. Refrigerated incubator set to 27 °C.

  3. Laminar flow hood (biosafety cabinet) for sterile work.

  4. Sf9 cells adapted to growth in suspension culture in serum-free insect cell media.

  5. Serum-free insect cell growth medium (e.g., Sf-900 II SFM).

  6. Pen-Strep solution: 10,000 IU penicillin, 10,000 μg/mL streptomycin in 100 mL of distilled water.

  7. P3 stock of recombinant baculovirus encoding the glycoprotein gene of interest (see Note 2).

  8. Trypan blue solution: 0.4% trypan blue (w/v) in PBS.

2.2. Protein Purification

  1. Assembled tangential flow filtration (TFF) system (see Note 3).

  2. 1-L bottle top 0.22 μm filters and 1- or 2-L compatible bottles.

  3. Empty 10-mL chromatography column (e.g., Bio-Rad PolyPrep® chromatography columns).

  4. Small peristaltic pump (capable of 0.1–20 mL/min flow rates).

  5. PBS buffer: phosphate-buffered saline pH 7.4.

  6. TN Buffer: 20 mM Tris-HCl pH 8.0, 150 mM NaCl.

  7. TNE Buffer: 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA.

  8. Phenylmethylsulfonyl fluoride (PMSF) solution: 100 mM PMSF in isopropanol (caution: cytotoxic, use eye protection).

  9. Superdex 200 10/300 GL column or a similar size-exclusion column.

  10. Amicon Ultra-15 50K concentrator (Millipore) or a similar concentrator.

  11. Amicon Ultra-4 50K concentrator (Millipore) or a similar concentrator.

  12. 0.1 μm PVDF centrifugal filter.

  13. Immunoaffinity column prepared by conjugating 10–15 mg of purified IgG to 1 mL of CNBr-Activated Sepharose™ 4B (GE Healthcare) or a similar resin following the manufacturer’s instructions and then packed into an empty 10-mL chromatography column. For purification of HSV-1 gB730, we typically use monoclonal antibody DL16 [32].

  14. AB-W buffer: 10 mM Tris-HCl pH 8.0, 500 mM NaCl.

  15. Potassium thiocyanate (KSCN) solution: 3 M KSCN in distilled water.

  16. Sodium azide solution: 0.025% sodium azide in distilled water.

  17. IMAC resin: Ni Sepharose Excel (GE Healthcare Life Sciences), Nickel Sepharose 6 Fast Flow (GE Healthcare Life Sciences), or a similar Ni resin (see Note 4).

  18. Nutating shaker.

  19. Ni-B buffer: 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 20 mM imidazole.

  20. Ni-W buffer: 20 mM Tris-HCl pH 8.0, 500 mM NaCl, 40 mM imidazole.

  21. Ni-E buffer: 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 300 mM imidazole.

  22. Imidazole stock solution: 4 M imidazole in distilled water.

  23. EDTA solution: 0.5 M EDTA pH 8.0 in distilled water.

2.3. Crystallization

  1. 24-well pregreased crystallization plates (Hampton Research).

  2. 22 mm siliconized circle cover slides (Hampton Research).

  3. Aerosol duster (Thermo Fisher Scientific) or in-house air.

  4. Stereomicroscope for crystal observation.

  5. gB crystallization solution: 15% PEG (polyethylene glycol) 4000 (w/v), 200 mM NaCl, 100 mM Na citrate pH 5.5, sterile filtered.

  6. gH/gL crystallization solution: 20% PEG 4000 (w/v), 100 mM Na citrate pH 4.5, sterile filtered.

  7. CryoLoops (Hampton Research).

  8. Cryo storage vials with compatible mounting bases (e.g., CrystalCap Copper Magnetic [Hampton Research]).

  9. CrystalWand handling tool and a vial clamp (Hampton Research).

  10. CryoCanes and CryoSleeves for storage of cryo storage vials (Hampton Research).

  11. 0% mesoerythritol gB cryopreservation (cryo) solution: 15% PEG 4000 (w/v), 350 mM NaCl, 100 mM Na citrate pH 5.5.

  12. 5% mesoerythritol gB cryo solution: 5% mesoerythritol (v/v), 15% PEG 4000 (w/v), 350 mM NaCl, 100 mM Na citrate pH 5.5.

  13. 10% mesoerythritol gB cryo solution: 10% mesoerythritol (v/v), 15% PEG 4000 (w/v), 350 mM NaCl, 100 mM Na citrate pH 5.5.

  14. 15% mesoerythritol gB cryo solution: 15% mesoerythritol (v/v), 15% PEG 4000 (w/v), 350 mM NaCl, 100 mM Na citrate pH 5.5.

  15. gH/gL cryo solution: 20% xylitol (w/v), 20% PEG 4000 (w/v), 150 mM NaCl, 100 mM Na citrate pH 4.5.

  16. Liquid nitrogen.

  17. Liquid nitrogen Dewar flasks for crystal freezing and temporary crystal cane storage (Hampton Research).

3. Methods

3.1. Protein Expression

  1. Wash, assemble, and autoclave 3-L spinner flasks and the 2-port airflow assemblies with 0.2 μm air filters as per the manufacturer’s instructions (see Note 5).

  2. Expand Sf9 cells to obtain appropriate volume of suspension culture in log phase at a density of 2 × 106 cells/mL using SF900II SFM plus Pen-Strep solution. The viability should be 97% or above as determined by trypan blue staining (see Note 6). Cell culture volume should be calculated based on the required amount of pure protein and the expected yield. 1–1.6-L cultures can be grown in a 3-L flask. Our typical yields are ~2 mg HSV-1 gB730 per liter of culture or 250 μg HSV-2 gH803-H6/gL per liter of culture.

  3. In a laminar flow biosafety cabinet, infect Sf9 cells with recombinant baculovirus by adding 4–10 mL of P3 per 1 L of cells (see Note 7).

  4. Incubate infected cells for 72 h at 27 °C, with a spinner setting of 75–90 rpm and with airflow adjusted to prevent excessive bubble accumulation (see Note 8).

3.2. Removal of Medium Components from Insect Cell Supernatants

  1. Harvest the cell culture supernatant containing secreted protein by centrifuging the cell suspension at 3725 × g for 35 min at 4 °C. Discard the pellet. For this and all subsequent steps, a sterile environment is not required.

  2. During the centrifugation step, begin flushing the TFF system with distilled water according to the manufacturer’s instructions. After flushing the TFF system with water, equilibrate the TFF filter with PBS for at least 15 min at 4 °C in a cold room (see Note 9).

  3. Vacuum filter the supernatant through a 1-L 0.22 μm bottle-top filter.

  4. Add PMSF solution to a final concentration of 0.1 mM to inhibit proteases. Take out a 50 μL aliquot of this sample to monitor protein loss during purification.

  5. Transfer the filtered supernatant to a cold room. All subsequent steps must be done at 4 °C to reduce protein degradation.

  6. Properly position the tubing of the TFF system prior to starting TFF. Place the end of the large feed flow tube into the bottle containing the supernatant; this bottle will act as the feed tank (Fig. 1). Thread the rest of the large feed flow tube through the Easy-Load peristaltic pump. Direct the end of the retentate tube (coming out of the edge of the top surface of the TFF filter) back into the feed tank. Direct the permeate tube into a waste container (Fig. 1). Turn on the pump and adjust the hosecock clamp on the retentate tube to achieve the desired pressure to drive filtration (see Note 10).

  7. Once the supernatant is reduced to the desired volume (we typically use 350–550 mL: 200–400 mL in the bottle and 150 mL in the tubing of the TFF system), begin buffer exchange (see Note 11).

  8. Set up Exponential Buffer Exchange: First measure the amount of permeate being produced, in mL/min. Then set up a small peristaltic pump to add the desired buffer (PBS for gB730, or TN for gH/gL) to the supernatant at the same rate that permeate is being removed. This is the quickest way to do buffer exchange but is not suitable for all preparations (see Note 12).

  9. Perform buffer exchange until the percentage of original media in the retentate has been reduced to the desired amount: 0.5% for gB730 and 0.2% for gH803-H6/gL (see Note 13).

  10. When finished concentrating the supernatant or performing buffer exchange, place the feed flow tubing in a bottle that contains 200–300 mL of PBS (the retentate tubing should remain in the feed tank). Loosen the hosecock clamp on the retentate tubing and pump the PBS through the TFF filter until the PBS has flowed through the TFF system but stopping before air gets pumped through the TFF filter. Do this even if buffer exchange is not performed to help remove residual protein that may be on the TFF filter.

  11. Add PMSF solution to a concentration of 0.1 mM and save 50 μL aliquots of the TFF retentate and permeate to monitor protein loss during purification.

  12. For gB and other proteins purified using immunoaffinity, go to Subheading 3.3. For gH/gL and other proteins purified by Ni IMAC, go to Subheading 3.4. Purification using other affinity tags will require protocol modification based on the affinity resin manufacturer’s instructions.

Fig. 1.

Fig. 1

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A schematic of the tangential flow filtration (TFF) setup described in Note 3. The supernatant is pumped into a pressurized filter. The filtrate (permeate) is removed, and the retentate is recirculated. Buffer can be added to replace lost medium. Arrows indicate the direction of the flow and the tubing

3.3. Immunoaffinity Purification

  1. Equilibrate the immunoaffinity column in AB-W buffer using a peristaltic pump. This and all subsequent purification steps are done at 4 °C.

  2. Load the TFF retentate onto the immunoaffinity column using a small peristaltic pump and adjust the flow rate such that the retentate will bind the column overnight. Save a 50 μL aliquot of the flow through to monitor protein loss during purification.

  3. Wash the column with ~70 column volumes (CV) of AB-W buffer to remove nonspecifically bound proteins and other molecules. This step should be optimized for optimal protein purity and yield. Save a 50 μL aliquot of the wash fraction.

  4. Add 20 μL of PMSF solution to an empty tube and elute into that tube at 1 mL/min with 20 CV of KSCN solution. Save a 50 μL aliquot of the eluate fraction. Concentrate the eluate to 1 mL or less in an Amicon-15 concentrator prior to further purification by size-exclusion chromatography. Go to Subheading 3.5 for the next step: size-exclusion chromatography.

  5. Wash the column with 75 CV of AB-W buffer plus sodium azide solution to prevent microbial growth and seal it for storage.

3.4. IMAC Purification

  1. To reduce nonspecific binding to the Ni resin, 20 mM imidazole should be added to the TFF retentate containing gH/gL (see Note 14). This step should be optimized for optimal protein purity and yield.

  2. Capture gH/gL on Ni resin in a batch-binding mode by incubating 0.5 mL of Ni resin with 45 mL TFF retentate in a 50 mL conical tube for 1 h at 4 °C on a nutating shaker (see Note 15). This and all subsequent steps are done at 4 °C.

  3. Recover the Ni resin containing bound gH/gL by centrifuging the 50 mL conical tube at 1000 × g for 5 min. Remove the supernatant and collect it in a container labeled flow through. Repeat steps 2 and 3 until all the TFF retentate containing gH/gL has been mixed with the Ni resin for binding. We recommend saving all flow through in this and subsequent steps for SDS-PAGE analysis.

  4. Resuspend the Ni resin with 5 mL Ni-B buffer in the 50 mL conical tube and then transfer it to an empty 10 mL chromatography column. Rinse the 50 mL conical tube with 2–3 mL of Ni-B buffer to collect residual Ni resin from the sides of the tube and add it to the chromatography column. Allow the Ni-B buffer to flow through the column and collect in container labeled wash 1. We recommend using gravity flow for gH/gL purification (see Note 16).

  5. Wash the column with 10 column volumes (CV) of Ni-B buffer (see Note 17) and continue to collect the flow through in the container labeled wash 1.

  6. Wash the column with 10 CV of Ni-W buffer and collect the flow through in a container label wash 2. This step is required for gH/gL to remove a 43 kDa cathepsin contaminant (see Note 18).

  7. Prepare a 15 mL conical tube containing 20 μL of PMSF solution and 100 μL of EDTA solution. Elute into this tube using 10 CV of Ni-E buffer (see Note 19). Imidazole concentrations and the volume of Ni-E buffer should be optimized. Concentrate the eluate to 1 mL or less in an Amicon-15 concentrator. Further purification is then done using size-exclusion chromatography, as described in Subheading 3.5.

3.5. Size-Exclusion Chromatography

  1. Load the concentrated protein purified by IMAC or immunoaffinity chromatography into a 0.1 μm PVDF centrifugal filter and centrifuge the protein at 17,000 × g and 4 °C for 10 min to remove any particulates. Load the protein onto a size-exclusion column (e.g., Superdex S200 10/30 GL) equilibrated in TNE buffer and collect the peak corresponding to monodisperse protein. gB730 elutes ~2.5 mL after the void volume while gH/gL elutes ~5.5 mL after the void volume on a Superdex S200 10/30 GL column.

  2. Concentrate the protein to 3.5–4.5 mg/mL (gB730) or 1.4–1.8 mg/mL (gH803-H6/gL) using an Amicon Ultra-4 concentrator. Concentrations of 4–6 mg/mL are recommended for initial crystallization trials on other glycoproteins.

3.6. Crystallization

  1. Use a pipette tip to make a notch in the grease ring around the edge of each well in a pre-greased 24-well plate.

  2. Add 750 μL of filtered crystallization solution to each well that will be used (see Note 20).

  3. Briefly spray a siliconized glass cover slip with air, and put it face up on a clean work surface. Add 1 μL crystallization solution and 1 μL protein to the center of the cover slip; then flip it over and use it to seal the well. Use the back of a 1 mL pipet tip to press the cover slip down evenly without smudging it. Repeat these steps with additional crystal setups.

  4. Store the crystal plate in a vibration-free environment at a constant temperature. gB crystals appear as hexagonal rods after 3–6 days and grow to their final sizes of 0.1–0.5 mm after 2–4 weeks. It is normal for some granular precipitate to appear within 48 h. gH/gL crystals are tetragonal and appear after 4–5 days, growing to their final size of 0.1–0.2 mm after 2–3 weeks (Fig. 2).

  5. Freezing gH/gL crystals. Working at the microscope, place a 2 μL drop of gH/gL cryo solution on a new siliconized glass cover slide. Carefully remove the cover slide with the crystal drop from the well and flip it over. Use a mounted cryoloop of appropriate size (slightly larger than the crystal) on the end of the long crystal wand to scoop up a crystal and place it in the drop of cryo solution briefly (10 s to 5 min), and then plunge the crystal into liquid nitrogen (see Note 21). Use the vial clamp to place the vial on the crystal cap without removing either from liquid nitrogen. Store vials long term in CryoCanes surrounded by cryosleeves in a liquid nitrogen Dewar flask or collect data on them immediately.

  6. Freezing gB crystals. gB crystals are not all grown under the same crystal conditions. The cryo solution for gB should match the well solution in which the crystals grew plus an extra 150 mM NaCl to account for NaCl present in the protein solution and 15% mesoerythritol as a cryoprotectant. gB requires stepwise addition of cryoprotectant, so cryo solutions containing 0%, 5%, 10%, and 15% mesoerythritol should be made prior to freezing crystals. Initially transfer the crystal into a 2 μL drop of 0% mesoerythritol gB cryo solution. Then add 2 μL of the 5% mesoerythritol gB cryo solution drop and remove 2 μL of liquid from the other side of the drop, watching carefully to avoid pipetting up the crystal. Repeat this with the 10% and 15% gB cryo solutions. Finally, transfer the crystal briefly to a drop of 15% mesoerythritol gB cryo solution, and then plunge into liquid nitrogen and proceed as described for gH/gL crystals.

Fig. 2.

Fig. 2

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(a) Elongated trigonal crystals of HSV-1 gB730. (b) a tetragonal crystal of HSV-2 Δ48gH803/gL

Acknowledgments

We acknowledge the contributions of the laboratory of Roselyn Eisenberg and Gary Cohen toward the development of the initial purification protocols of HSV-1 gB730 and HSV-2 gH803/gL produced using recombinant baculoviruses. We also thank Tirumala K. Chowdary and Sapna Sharma for their work in establishing and optimizing these protocols in our laboratory. Finally, we thank past and present members of the Heldwein lab for helpful advice and discussions.

4 Notes

1.

Use of aeration is necessary for growing insect cells in volumes of 1 L and above. Insect cells can also be grown in shaker flasks, but that method is not described here.

2.

The recombinant baculovirus used for gB730 expression encodes HSV-1 gB residues 31-730 with a melittin signal sequence, described previously [17, 32]. The HSV-2 Δ48gH803-His6/gL construct used for crystallization encodes all of gL and the N-terminally truncated gH ectodomain 48-803, as well as the gH N-terminal signal sequence, residues 1-18, also described previously [23].

3.

Several complete TFF systems are available on the market. Nevertheless, due to its reliability and low cost, we recommend assembling a basic TFF system from separate components, which include an Easy-Load Peristaltic Pump (Millipore XX80EL000) with dedicated 3/8″ inner diameter tubing (Millipore XX802GS25), a pressure gauge (Millipore YY1301015) with fittings for 5/16” inner diameter tubing, a Prep/Scale 30 kDa TFF Filter (Millipore), a ring stand and clamps to hold the system in place, 3/16” inner diameter tubing (Millipore XX8000T24) (for permeate), 5/16” inner diameter tubing (for retentate), fuel hose clamps, and a hosecock clamp (Fisher 05-847Q). The TFF system should be assembled as follows: Connect 4 ft of the large feed flow tubing used for the Easy-Flow Pump (3/8” inner diameter) to the pressure gauge via the fittings. Seal the connection by wrapping the connected end of the tubing (over the fitting) with two layers of paper towel and tightening a fuel hose clamp around the paper towel. Repeat this method to connect the other end of the pressure gauge to the feed port on the bottom of the TFF filter; use paper towel plus fuel hose clamps to tighten the tubing at both the fitting on the pressure gauge and at the feed port on the TFF filter. Connect ~3 ft of 5/16” diameter tubing to the retentate port on the TFF filter; connect ~4 ft of 3/16” tubing to the permeate port in the middle of the top of the TFF filter. Place a hosecock clamp on the retentate tubing near its connection to the TFF filter. Flush with distilled water at 20 psi for at least 2 min to ensure that there are no leaks (loosen or tighten fuel hose clamps to eliminate leaks). See Fig. 1 and the TFF filter documentation for additional details.

4.

Use of the Ni Sepharose Excel resin eliminates the need for buffer exchange before binding the protein to the Ni resin because this resin is more resistant to being stripped by the metal-chelating agents in the cell media, so the filtered supernatant can be applied directly to the resin.

5.

Spinner flasks should be autoclaved twice. For the first autoclave cycle, use a 20 min liquid cycle without attaching air filters and with ~1” of distilled water at the bottom of the flask. After the first cycle, empty the water from the flask and attach the air filters to airflow ports on the flask. Then cover all caps, airflow ports, and filters on the flask with aluminum foil. For the second autoclave cycle, use a 30- or 40 min gravity cycle.

6.

Take a 1 mL sample of the insect cell suspension culture. Mix 10 μL of the insect cell suspension culture sample with 10 μL of trypan blue solution to make a 1:1 dilution of the cell suspension in trypan blue. Load 10 μL of the cell suspension with the trypan blue onto a hemocytometer. Cells that appear blue are dead and cells that appear white are living. Count the living and the dead cells (separately) in one 1 × 1 mm square of the hemocytometer. The viability can be determined by dividing the number of living cells by the total number of cells.

7.

It is not necessary to determine the titer of baculovirus stocks used for protein expression because the amount of baculovirus necessary to achieve optimal protein expression is best determined experimentally. In our experience, using 4–10 mL of P3 stock per 1 L cells at a density 2 × 106 cells/mL is typically a good starting point regardless of the glycoprotein nature and often does not require further optimization. During optimization of protein expression, we find it helpful to monitor cells death during expression. If excessive cell death, viability of 60% or less, is observed after 3 days, the amount of P3 used for infection should be decreased. If protein expression is low, the amount of P3 used for infection should be increased. Volumes within a 1–20 mL range have been used successfully.

8.

Air settings of 25–50 mL/min are often used for 1 L cultures and of 50–100 mL/min for 1.6 L cultures, but airflow should be adjusted to minimize frothing on the surface of the culture (no more than a thin layer of bubbles and should not appear foamy).

9.

The TFF system may be flushed with distilled water at either room temperature or at 4 °C. However, it is ideal to equilibrate the system with PBS at 4 °C. To equilibrate the TFF system in PBS the inlet tubing, waste tubing, and retentate tubing all go into the “feed tank” bottle of 400 mL of PBS so that the PBS is recycled as it flows through the system. Refer to Fig. 1.

10.

We recommend using the maximum pressure that will not damage the filter. We use 10 psi. Higher pressures provide faster filtration but may promote protein aggregation.

11.

Buffer exchange only needs to be done for immunoaffinity purification or if using standard Ni resin for purification. Longer time for TFF and buffer exchange may lead to protein aggregation on the TFF filter.

12.

Exponential buffer exchange works quickly because, at every moment, the highest possible fraction of supernatant is replaced by buffer. However, it is done at low volume (high protein concentration) and high pressure; some proteins may aggregate under these conditions. In that case, either try exponential buffer exchange at a higher static volume (500 mL or more instead of 350 mL) or do buffer exchange by iterative dilution: pouring in 1 L buffer every time the total volume drops to the desired low point (350 mL, which is 200 mL in the bottle and 150 mL in the TFF system).

13.

For exponential buffer exchange, the percent of original media components still resent in the retentate can be calculated by using the “continuously compounded interest” equation b=ae(−rv)t, where b is the current percent of original media, a is the original percent (100, unless some buffer exchange was done previously), r is the permeate flow rate (mL/min), v is the retentate volume, and t is the time (minutes). For iterative dilution buffer exchange (see Note 12), the equation used for each dilution is = aVcVd, where b is the current percent of original media after the retentate has been diluted, a is the percent prior to dilution, and Vc and Vd are the volumes of retentate both before and after dilution (which includes the approximately 150 mL of retentate circulating through the TFF system). We find that 0.5% works well for gB and recommend this value for other proteins purified using immunoaffinity chromatography. gH/gL requires more extensive TFF, to 0.2% or less original media, due to presence of cobalt in the insect cell medium possibly interfering with binding of the His6-tag on gH/gL to Ni resin. This step should be optimized for different constructs, and especially for different affinity tags. Contact the manufacturer of the insect cell medium to find out which components may interfere with the specific affinity purification.

14.

For some His-tagged proteins, addition of 10 mM imidazole or leaving out imidazole helps improve binding.

15.

Batch binding is required for effective binding of gH/gL to Ni resin. Several 50 mL conical tubes with Ni resin may be used to speed up the binding process. Other proteins may efficiently bind Ni resin using gravity or peristaltic pump-aided flow.

16.

For gH/gL, yield of monodisperse protein is improved slightly when using gravity flow instead of pumps. Peristaltic flow at 2 mL/min can be used to accelerate purification during all steps and typically works well with other proteins.

17.

Longer wash with Ni-B buffer may be necessary depending on the preparation size. The amount of washing and imidazole concentration may need to be optimized for other proteins. For His-tagged proteins other than gH/gL, we recommend starting with a 10 mM imidazole concentration in the initial wash buffer.

18.

Longer wash with Ni-W buffer may be necessary. Imidazole concentration, salt concentration, and the amount of washing may need to be optimized for other proteins. For His-tagged proteins other than gH/gL, we recommend starting with a 20 mM imidazole and 100–150 mM NaCl in the Ni-W buffer.

19.

To increase gH/gL purity, 140 mM imidazole can be used during the elution step because several contaminants bind Ni resin more tightly than gH/gL.

20.

gH/gL crystallizes with 20% PEG 4000 and 100 mM Na citrate, pH 4.5. Optimization of these conditions did not improve crystals in our experience. Crystal reproducibility is low, and we recommend setting up multiple identical crystallization drops for gH/gL. gB crystallizes under multiple conditions [17, 27, 28] but most easily under 15% PEG 4000, 200 mM NaCl, and 100 mM Na citrate, pH 5.5. Larger crystals can often be obtained by reducing PEG 4000 concentration to 10% or lower. gB crystallization can be optimized by a grid screen of 0.1, 0.2, 0.3, 0.4, and 0.5 M NaCl versus 1% increments of PEG 4000 from 6% to 15%.

21.

Crystal drops will begin to dry out once exposed to air. 2 μL of well solution may be added to the drop to prevent crystal degradation short-term, up to 30 min, although doing so may compromise the drop if the coverslip is resealed onto the well for later use.

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