Expression of HA NA using Baculovoirus

Generation of Recombinant Baculovirus

Cloning into baculovirus transfer-plasmids

Clone H7 HA and N9 NA ectodomain (or any other HA or NA gene) into modified pFastBac transfer plasmids (see introduction). Vectors for HA containing a C-terminal trimerization domain and a hexahistidine tag, as well as for NA containing an N-terminal tetramerization domain and hexahistidine tag

Explanatory note: HA ectodomains expressed without a trimerization domain will be folded incorrectly, leading to loss of conformational neutralizing epitopes, especially in the stalk domain. Similarly, the NA enzymatic function relies on proper tetramerization.

Generation of bacmids

Transform baculovirus transfer-plasmids containing HA and NA genes into DH10Bac bacteria according using a baculovirus expression system per manufacturer's instructions. Isolate and purify bacmids using a mini or midiprep kit.

Rescue of recombinant baculovirus and verification of protein expression

Explanatory note: The following steps have to be performed aseptically in a laminar flow hood Perform this procedure independently for HA and NA (or any other gene of interest).

Insect cells are usually grown at 28 °C without CO2. Sf9 cells for this protocol are grown in full TNM-FH insect cell media supplemented with 10% FBS, 1% Pen-Strep* and 0.1% Pluronic F68 solution, if not indicated otherwise, and passaged 1:4 twice a week. High Five cells are grown in SFX serum free media and passaged 1:15 twice a week.

• Addition of antibiotics should be considered optional throughout the protocol and adjusted according to the practices used in each laboratory.
  
  

1. Plate Sf9 insect cells in 6-well plates at a density of 2 x 105 cells/cm2 and let sit for 20 min in a 28 °C incubator (without CO2).

2. Mix 2 μg (conc. 0.5-2 μg/ul) of recombinant bacmid with 100 μl of TNM-FH medium (serum free, 1% Pen-Strep). Mix 6 μl of transfection agent with 100 μl of TNM-FH medium (serum free, Pen-Strep). Combine the two volumes, mix gently and let sit for 20 min at room temperature (transfection mixture). Include one mock-transfection as control.

3. Remove supernatant from plated Sf9 cells (cells will now stick to the plate) and replace with 2 ml of serum free TNM-FH medium containing Pen-Strep (1%). Add the complete volume of the transfection mixture (from step 1.2.2) and rock the 6-well plate carefully for 5 sec. Incubate in a 28 °C incubator without CO2 for 6 hr.

4. Replace media with fresh full TNM-FH media (containing 10% FBS, 1% Pen-Strep and 0.1% Pluronic F68). Incubate in a 28 °C incubator without CO2 for 6 days. Check cells occasionally under a microscope. Infected cells usually appear bigger, rounder, have enlarged nuclei, and start to detach.

Harvest of cells and confirmation of protein expression

1. Harvest cells 6 days post transfection by centrifugation at 2,000 x g for 5 min at room temperature. The supernatant contains the recombinant baculovirus - this will be used to generate working stocks right away, or can be stored at 4 °C for years.

2. To check protein expression collect pellets and resuspend in 500 μl of PBS. Mix 50 μl of the suspension with 50 μl of SDS-PAGE loading buffer (containing a reducing agent). Run SDS-PAGE and perform a Western blot analysis with an anti-hexahistidine antibody to confirm gene expression. Include cells from the mock transfection as negative controls.

Preparation of working stocks

1. Plate 5 x 105 Sf9 cells/cm2 in a T175 cm2 flask and let sit for 20 min allow them to attach to the dish. Replace complete TNM-FH media with TNM-FH media containing 3% FBS (and 1% Pen-Strep, 0.1% Pluronic F68).

2. Infect cells with 100 μl of the rescue supernatant. Incubate in a 28 °C incubator without CO2 for 6 days and check occasionally if cells get infected.

3. Spin at 2,000 x g at room temperature for 5 min and collect supernatant. This is now your P2 stock. This stock can be stored indefinitely at 4 °C. Repeat the infection procedure using 100 μl of the P2 stock to infect cells. The resulting stock is called P3 or working stock, which can be stored at 4 °C or used directly for protein expression. The P3 stock usually contains 107-109 plaque forming units/ml.

Protein Expression and Purification

Explanatory note: Sf9 cells support the growth of baculovirus very well. However, their capacity to secrete large amounts of recombinant protein is limited. Therefore  High Five  insect cell line should be used for expression of secreted recombinant proteins. These cells do not support baculovirus replication to very high titers but have high secretory capacity.

Growing up High Five cells

Grow High Five cells in SFX insect cell culture medium in T175 cm2 flasks. Passage every other day 1:3. For a 200 ml expression culture you will need to grow 5 confluent flasks.

Harvesting and infecting High Five cells

1. Detach High Five cells from 5 T175 cm2 flasks by tapping the flasks with your hand. Collect the cell suspension and transfer into 50 ml centrifugation tubes.
2. Spin at 1,200 x g for 7 min at room temperature. Remove supernatant by decanting and unite the pellets by resuspending them in 15 ml of P3 stock.
3. Let sit for 15 min in the flow hood (Figure 5).
4. Transfer 200 ml of Hyclone SFX culture medium into a clean, sterile 1,000 ml shaker flask (without buffles, sealed with aluminium foil before autoclaving, should not have been used for bacterial cultures before).
5. Transfer the P3/cell suspension into the shaker flask. Seal with sterile aluminium foil and transfer flask into a shaker. Shake at 70 rpm at 28 °C for 72-96 hr.

Harvesting recombinant protein from High Five cell expression supernatants

1. 72-96 hr post infection transfer culture supernatants into 500 ml centrifugation buckets. Spin at 5,500 x g for 20 min at 4 °C. In the meantime rinse the shaker flasks 3x with double distilled water (ddH2O).

2. Transfer 3 ml Ni-resin slurry into a 50 ml tube with 45 ml of PBS (one needed per 200 ml culture). Shake well and spin at 3,000 x g at room temperature for 10 min. Decant the PBS and keep the pellet Mix the insect cell supernatant with the Ni-resin pellet and transfer into the already rinsed shaker flasks. Incubate at 4 °C for 2-4 hr, while shaking.

3. Mount 10 ml polypropylene column on a clamp on a support stand. Remove both caps and place a beaker below the column to collect flow through (waste). Take the shaker flask out of the shaker and start to transfer the supernatant/slurry mixture onto the column; it will retain the Ni-resin and only the supernatant will flow through. Pass the entire volume over the column.

4. Wash the resin 4x with 15 ml of washing buffer (50 mM Na2HCO3, 300 mM NaCl, 20 mM imidazole, pH 8). Let all the washing buffer drain from the column and close it with the cap (lower side).

5. Add 2 ml of elution buffer (50 mM Na2HCO3, 300 mM NaCl, 300 mM imidazole, pH 8) and let sit for 5 min. Open column and collect the eluate. Repeat this 3 more times (with a total of 8 ml of elution buffer). Eluate that contains high concentrations of protein typically shows foam when shaken. Keep eluate on wet ice whenever possible.

Buffer exchange and concentration

1. Prespin 15 ml ultrafiltration centrifugation units (30 kDa cut-off for HA/NA but may vary depending on the purified protein size) with PBS (pH 7.4-7.6) at 3,000 x g at 4 °C for 20 min. Discard flow through and load eluate onto the spin column Fill up with 5 ml of sterile, ice cold PBS and spin for 60 min at 3,000 x g at 4 °C.

2. Discharge flow through again, refill with 15 ml of ice cold PBS and spin for 60 min at 3,000 x g at 4 °C. Repeat this procedure one more time.

3. Take the spin column out of the centrifuge and collect the buffer exchanged concentrate (typically 200 μl). This is highly concentrated recombinant protein. Rinse the spin column with 400 μl of sterile ice cold PBS and add this to the concentrate. Keep the concentrate on ice at all times.

Characterization and Quality Control

1. Measuring protein concentration
Take an aliquot of the concentrate and measure it by using your protein quantification method of choice. Set protein concentration to 1 mg/ml by adding PBS and freeze small aliquots to -80 °C. Freeze thaw cycles harm the protein structure and can lead to aggregation.

2. SDS-PAGE and Coomassie staining
Run 500 ng of your protein on a SDS-PAGE and perform a Coomassie staining. For fast and convenient staining use Coomassie reagent in combination with a microwave protocol. HA proteins typically appear at 64 kDa whereas NA proteins should run at a size of around 56 kDa. Using the described protocol you should not see any degradation products. If degradation occurs it is usually a sign of yeast/fungal contamination during expression. In this case repeat the procedure and make sure to keep everything sterile.

3. Western blot/ELISA
In order to verify protein identity we recommend do to a Western blot or ELISA assay with a specific antibody. Ideally, ELISA assays are performed with an antibody that binds to conformational epitopes (e.g. anti-HA stalk antibodies1). Using such an antibody the correct folding of the protein can be confirmed.

4. Measuring NA activity
Correct folding of the viral NA can also be determined by measuring NA activity. We recommend to perform this test using the commercially available NA*STAR assay.