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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 : )