Purification of Viral Integrase Using Heparin Affinity Chromatography

Begin with a purified viral integrase enzyme fraction containing residual bacterial nuclease as a contaminant.

Load the sample onto a heparin-Sepharose column pre-equilibrated with a low-salt binding buffer.

As the mixture passes through the column, the integrase binds more strongly to the heparin ligand than the contaminating nuclease.

Next, gradually increase the salt concentration in the buffer flowing through the column.

At low salt concentrations, weakly bound nucleases elute first, while strongly bound integrase elutes later at higher salt concentrations.

Collect the eluted fractions individually to separate the contaminating nuclease from the integrase.

Add SDS-PAGE sample buffer to each fraction and heat to denature the proteins. Load the samples onto an SDS-PAGE gel and perform electrophoretic separation.

Observe the stained gel.

Identify the fractions containing a prominent integrase band with no visible contaminating proteins, indicating apparent purity.

To perform heparin affinity chromatography fractionation of the prototype foamy virus integrase, reduce the sodium chloride concentration of the sample to 200 millimolar by adding 1.5 volumes of buffer C. Then load the diluted pr