LABORATORY 6: PURIFYING THE FLUORESCENT PROTEIN

1. LABORATORY 6: PURIFYING THE FLUORESCENT PROTEIN 2014

2. Overview • In Laboratory 5, students incorporated recombinant plasmid with rfp gene into a living cell and confirmed that transformation was successful • In Laboratory 6, students will obtain cell proteins and purify red fluorescent protein from transformed cells grown in shaker flask overnight

3. PART A: Lyse bacteria cells

4. Reasons for lysis • Soluble proteins made by cell, including red fluorescent protein, are dissolved in cell cytoplasm • Only way to access soluble proteins is to lyse (break open) cell • After lysis, soluble proteins can be easily separated from insoluble structural proteins

5. Procedure • Obtain 1mL cell culture in tube • Centrifuge for 5 min and identify location of red fluorescent protein • Remove supernatant with pipette • Add another 1mL of cell culture to tube and centrifuge for 5 min • Remove supernatant with pipette

6. Procedure • Tip tube and remove last bit of supernatant with pipette, without touching cell pellet • Add elution buffer and drag closed tube across tube rack to resuspend cells • Add lysis buffer and incubate cells overnight at room temperature to release proteins from the cells

7. PART B: Separate RFP

8. Reasons for separation • Although the bacteria make a lot of red fluorescent protein, there are up to 1,000 other proteins in a living cell • Those other proteins might interfere with intended use of RFP or of any other protein you are isolating • Pharmaceutical companies require purified protein

9. Procedure • Centrifuge cells (be sure to balance), and remove supernatant with RFP • Add binding buffer to the supernatant • Add supernatant to column and drain • Add wash buffer to column and drain • Add elution buffer to column and collect RFP

10. Procedure • Always add liquids to the side of column • Never touch the resin with pipette • Drain until 2 mm of liquid is left above the column and never expose the resin to air • Wait until each solution has drained before adding another solution

11. Separation uses protein folding Unfolded Folded− hydrophobic areas covered

12. Amino acids’ electric charge • Proteins have hydrophobic and hydrophilic amino acids • Difference is electric charge distribution • Hydrophobic amino acids ARE NOT attracted to water molecules • Hydrophilic proteins ARE attracted to water molecules

13. Protein folding in binding buffer • In binding buffer, hydrophobic proteins unfold • Unfolded hydrophobic proteins adhere to the hydrophobic column resin • Folded hydrophilic proteins never adhere to the column Hydrophilic proteins

14. Protein folding in wash buffer • In wash buffer, moderately hydrophobic proteins fold • Highly hydrophobic proteins, including RFP, stay unfolded • Folded moderately hydrophobic proteins are released from the column Moderately hydrophobic proteins

15. Protein folding in elution buffer • In elution buffer, highly hydrophobic proteins, including RFP, fold • Folded highly hydrophobic proteins, including RFP, are released from the column • RFP can be collected RFP

16. Results