Heparin Affinity Chromatography-Based Baculovirus Purification: A Technique to Isolate Baculovirus From Insect Cell Supernatant

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

1. 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.
2. 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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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