Editing Expression of HA NA using Baculovoirus (section)

== Protein Expression and Purification ==

<br>

Explanatory note: Sf9 cells support the growth of baculovirus very well. However, their capacity to secrete large amounts of recombinant protein is limited. Therefore&nbsp; High Five&nbsp; 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 ===

<br> 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.<br> 

=== Harvesting and infecting High Five cells<br>  ===

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.<br> 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.<br> 3. Let sit for 15 min in the flow hood (Figure 5).<br> 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).<br> 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.

=== <br> Harvesting recombinant protein from High Five cell expression supernatants ===

<br> 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).

<br> 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.

<br> 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.

<br> 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).

<br> 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.

=== <br> Buffer exchange and concentration ===

<br> 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.

<br> 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.

<br> 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.