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 sodi…

Divulgazioni

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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Citazione di questo articolo
Heparin Affinity Chromatography-Based Baculovirus Purification: A Technique to Isolate Baculovirus From Insect Cell Supernatant. J. Vis. Exp. (Pending Publication), e21095, doi: (2023).

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