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 0.6mg of reflectin from the column. 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 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], to see if we could observe behaviour that we would expect from reflectin in this way. They produced some very interesting results!