LABORATORY 6PART B

PURIFICATION OF MFPFROM AN OVERNIGHT CULTURE

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

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

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

Separation uses protein folding

Unfolded Folded−

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

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

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

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

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

WHAT WILL YOU NEED TO DO?

• Preparation day 1 – lysing the cells• Preparation day 2 – mFP purification using column

chromatography

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

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

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

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!!!!

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!!

Purification of RFP from an overnight culture

Overnightculture

Cell pelletwith RFP

Lysedcells

Pelletcell debris

RFP withbinding buffer

Bruce Wallace

Results

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!!

IN SUMMARY:• Rfp genemfp 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 : )