Heparin Affinity Chromatography-Based Baculovirus Purification: A Technique to Isolate Baculovirus From Insect Cell Supernatant
成績單
Baculoviruses – double-stranded DNA viruses – are surrounded by an envelope containing major viral glycoproteins, like GP64.
To isolate baculoviruses from a cell culture supernatant containing cellular debris and contaminating proteins, assemble a heparin affinity chromatography column containing a matrix of cross-linked agarose beads. These beads are coupled to heparin, a linear polysaccharide with sulfate groups, which impart a negative charge to the heparin chain.
Equilibrate the column with a wash buffer having neutral pH and optimal ionic strength to provide favorable conditions for maximum baculovirus and heparin interactions during supernatant loading. Load the baculovirus-containing supernatant onto the column.
The basic amino acids in baculovirus GP64 facilitate nonspecific electrostatic interactions with the negatively charged heparin and bind tightly to the column, unlike contaminants which bind loosely. Rinse the column with a wash buffer, removing unbound and loosely bound contaminating proteins and debris.
Pass an elution buffer with higher ionic strength than wash buffer through the column. The higher ionic strength disrupts the baculovirus-heparin electrostatic interactions, dissociating and eluting baculoviruses from the column.
Collect the baculovirus-containing eluate in the flow-through. Immediately dilute the baculovirus suspension with a buffer containing physiological salt concentrations, preventing virus inactivation. The isolated baculoviruses can be used for further analysis.
