Heparin Affinity Chromatography-Based Baculovirus Purification: A Technique to Isolate Baculovirus From Insect Cell Supernatant
Published: April 30, 2023
Abstract
Source: Nasimuzzaman, M. et al., Production and Purification of Baculovirus for Gene Therapy Application. J. Vis. Exp. (2018)
In this video, we demonstrate the technique to isolate baculovirus from an insect cell supernatant using heparin affinity chromatography. The isolation is facilitated by the affinity between the heparin in the column to the baculovirus envelope glycoprotein.
Protocol
1. Preparation of the Chromatography System
- Prepare the chromatography system by sequentially rinsing the sample and buffer lines each with sterile water, 1 N sodium hydroxide, water, and wash buffer (20 mM sodium phosphate buffer containing 150 mM sodium chloride, pH 7.0) at a linear flow rate of 50 mL/min.
- Prepare a heparin 50 µm column (10 × 10 cm, 7.9 mL of column volume (CV)) by sequentially running column cleaning buffer (5 CV sterile 20 mM sodium phosphate buffer containing 2 M sodium chloride, pH 7.0) and 5 CV wash buffer at a linear flow rate of 7 mL/min.
2. Purification of Baculovirus Vector
- Set up the chromatography system as follows:
- Use the sample loading inlet port for loading the baculovirus supernatant.
- Use the buffer inlet port A1 for loading the wash buffer. Prepare a bottle with at least 500 mL of wash buffer and place the wash buffer line into the bottle.
- Use the buffer inlet port A2 for loading the elution buffer (20 mM sodium phosphate with 1.5 M sodium chloride, pH 8.0). Prepare a bottle with at least 500 mL of elution buffer and place the elution buffer line into the bottle.
- Insert several 50 mL conical tubes into the fraction collector to collect the column pass-through baculovirus supernatant, the wash buffer, and the eluted baculovirus.
- Equilibrate the heparin column with five 7.9 mL column volumes (CVs) of wash buffer (40 mL) at a linear flow rate of 7.0 mL/min.
- Load 250 mL of baculovirus supernatant onto the heparin column using the sample pump (inlet sample port) of the chromatography system at a linear flow rate of 2.0 mL/min.
- Run 10 CVs (80 mL) of wash buffer through heparin column at a linear flow rate of 2.0 mL/min until the ultraviolet (UV) absorbance curve (280 nm) has returned to baseline and becomes stable.
- Elute the baculovirus particles from the 7.9 mL heparin column with 5 CVs (40 mL) of elution buffer at a linear flow rate of 4.0 mL/min. Watch for a sharp elution peak of protein on the chromatogram when the baculovirus particles dissociate from the heparin column.
- Post-elution, immediately dilute the eluted baculovirus supernatant 10-fold using 20 mM sodium phosphate buffer in water to prevent inactivation of baculovirus particles from osmotic shock during subsequent centrifugation.
Declarações
The authors have nothing to disclose.
Materials
| Akta Avant 150 | GE Healthcare | 28976337 | Chromatography system |
| POROS Heparin 50 µm Column | ThermoFisher Scientific | 4333414 | Heparin column |
| Wash buffer | In-house | Non-catalog item | 20 mmol/l phosphate buffer containing 150 mmol/l sodium chloride |
| Elution buffer | In-house | Non-catalog item | 20 mmol/l phosphate buffer containing 1.5 mol/l sodium chloride |
| 50 ml Conical tube | ThermoFisher Scientific | 14-959-49A | For collection of Baculovirus supernatant |
| Column cleaning buffer | In-house | Non-catalog item | 20 mmol/l phosphate buffer containing 2.0 mol/l sodium chloride |
| Sterile water | In-house | Non-catalog item | For Akta Avant cleaning |
| Sodium hydroxide | Sigma-Aldrich | 1.09137 | For Akta Avant cleaning |
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Heparin Affinity Chromatography-Based Baculovirus Purification: A Technique to Isolate Baculovirus From Insect Cell Supernatant. J. Vis. Exp. (Pending Publication), e21095, doi: (2023).