Protein Purification

Bacteria expressing his-tagged reflectin were lysed, and the protein was purified using a his-trap column and a denaturing protocol in order to solubilise reflectin.

Practice

• After high copy expression plasmids for his-tagged reflectin were successfully assembled and transformed in E. coli, cell cultures were incubated overnight with 1mM arabinose to induce reflectin expression.
• Buffers were prepared, and their pH checked and readjusted if necessary on the day of purification.
• An inclusion body prep was performed with 50ml of overnight culture. Reflectin was purified from the resulting lysate using a his-trap column.
• This procedure was repeated using a culture of the same bacteria from a different flask.

Results

Photospectroscopy readings from the eluted solution indicated that we had obtained approximately 0.4mg of reflectin from the column for the denser culture, and 0.2mg from the other. After dialysis, a vacuum centrifuge was used to precipitate the protein. this precipitate was taken to the Nanophotonics centre in order to make our first reflectin thin films, and SDS-PAGE was run by another member of the department to check whether or not reflectin was contained in the precipitate. Unfortunately, no reflectin was identified in the gel, so we could not concretely verify that our protein purification protocol had been successful this way. We did not lose all hope, however, since other members of the lab assured us that they had previously received false negatives from SDS-PAGE, and we suspect that the quantity of protein produced was too low to detect. Hence, we pressed on with our [thin films experiment], which produced some very interesting results!

Since this was the first time any of us had tried to perform the his-trap technique, we also learnt some valuable lessons from our experiences in this first experiment. Some helpful 'tips' have been added to the his-trap purification protocol as a result, which really helped us to improve our yield of reflectin using this method.