1. LABORATORY 6 PART B PURIFICATION OF M FP FROM AN OVERNIGHT CULTURE

2. OVERVIEW: Goals: Explain confirmation of protein relates to function of protein How does protein folding occur? Lab: Take cells growing in broth: Lyse (break open) From overnight LB/amp/ara culture Purify mFP from cell lysate using column chromatography

3. INTRODUCTION Once laboratories locate promising therapeutic protein: Then locate and isolate gene that encodes the protein Insert gene into plasmid (to clone gene) Cloning vectors Plasmid engineered to replicate in high numbers Within bacterial cell Expression vectors pARA-R with rfp gene Plasmid engineered specifically for protein expression Transformed cells Allowed to express protein Lysed to release synthesized protein from cell

4. INTRODUCTION Mutant fluorescent protein: 238 aa in size Fluorophore located in center Highly hydrophobic In order to purify (separate) protein: Look for differences in hydrophobicity Hydrophobic verse hydrophilic Some have regions that are both Hydrophobic regions will “hide” in interior of molecule How to isolate a single protein? E. coli we are using produces HIGH concentrations of mFP

6. Separation uses protein folding Unfolded Folded−

7. INTRODUCTION Column chromatography Purification technique uses hydrophobicity to separate and purify proteins Plastic cylinder with resin Separating medium Contains small hydrophobic beads If mFP placed into solution of high salt concentrations: mFP molecule distorted Hydrophobic regions adhere to resin Hydrophilic proteins then continue down column and flushed away

8. INTRODUCTION mFP trapped in resin bed: Wash column with solution low salt concentration Hydrophobic regions of mFP point towards interior of molecule Will elute (wash out) moderately hydrophobic molecules with buffer Use solution of very low salt concentration to release mFP from resin beads

9. 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

10. 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

11. 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

12. WHAT WILL YOU NEED TO DO? Preparation day 1 – lysing the cells Preparation day 2 – mFP purification using column chromatography

13. 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

14. 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

15. WHAT WILL YOU NEED TO DO? Chromatography columns Capped tightly Stopcocks closed Store upright to allow resin bed to form flat surface Use ethanol to rinse resin if splashed on sides Open stopcock and let ethanol drain from column Leave about 2mm layer above resin bed

16. WHAT WILL YOU NEED TO DO? Chromatography columns Columns will have equilibration buffer (add 3000 µl equil. Buffer) Dispense down to 1 cm above resin Set up on ring stand for model High enough for collection below Make sure resin bed visible When done: Flush columns with 4-5 mL elution buffer Flush columns with 3 mL 20% ethanol Cap tightly!!!!

17. METHODS Do not let the supernatant (red) run through the tube….once it is gone, it is gone!! as soon as the red hits the first level of the chromatography column tip, get the 1.5 mL tube ready…collect when the red drips!!

18. Purification of RFP from an overnight culture Overnight culture Cell pellet with RFP Lysed cells Pellet cell debris RFP with binding buffer Bruce Wallace

19. Results

20. CONCLUSIONS What product do you have? Would this normally be the end? What's next?? Now that have purified protein, run steps to be sure it is purified Quality control, SDS Page, ELISA, Western Blot “fill and finish” Make into final form ready for distribution Ex: Enbr Two 20000 L tanks with bacteria Two 2L purified protein $2 million a Liter!!

21. IN SUMMARY: Rfp gene  mfp protein (highly hydrophobic) BB (Binding Buffer)- Unfolds hydrophobic mfp causing it to adhere to the resin. Washes out all other very hydrophilic elements WB (Wash Buffer)- Washes out the binding buffer and folded moderately hydrophobic proteins. EB (Elution Buffer)- Folds highly hydrophobic proteins (mfp) and releases them from the column last before washing out any other remaining elements. Leaves you with just the purified mfp: red flourescent protein, which glowed under UV light : )