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...