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Experiment 3: Purification of Fumarase
• Three week experiment
– Wraps up with formal lab report
• Abstract/Intro/Results/Discussion would be difficult
to write super early
• Keep up with M & M
• Think about/work on intro?
– DAQs after week 1
– Week 3 “DAQs” in lab report
• Protein Explorer exercise
– Assignment tomorrow
– Due date will be pushed back a couple of
days (originally Friday, March 16th)
• Grading
– Exams tomorrow
– Paper summaries
• Not even close
Why purify a protein?
• Determine amino acid sequence (1° structure)
• Determine 3-D structure (2°, 3°, 4°)
• Function of protein away from cellular partners
(in vitro)
– Interactions with other molecules
– Enzyme catalysis
– Inhibitors (drugs?)
Four ‘goals’ this week
• Lyse yeast
– Determine how much fumarase you start with
• Set up Nickel column
• Set up/become familiar with pump/fraction
collector
• Anion exchange ‘trial’
1st step of protein purification
Lysis
Centrifugation
Get rid of insoluble cellular components
-cell wall/membrane
-organelles
Soluble proteins
How are cells/tissues lysed?
• Enzymatic/chemical
– Degrade cell membrane
– Detergents/lytic enzymes
• Mechanical
– eg. Sonication (high energy sound waves)
Yeast are very tough: cell wall
• Mechanical lysis: Bead mill
– Yeast + Glass beads + “Blender”
– Lysis buffer
• Bit of detergent to weaken the cell (& stabilize
protein)
• Phosphate at ~ neutral pH, some salt
• Protease inhibitors
Purify/separate fumarase
• Physical/chemical characteristics: make it
‘unique’ among cellular proteins
• Here: we’ve altered the characteristics of
fumarase
– Genetic manipulation
– Add a ligand binding site…affinity purification
Purify/separate fumarase
Genomic (native) gene
for fumarase (DNA)
Codes for fumarase
(protein)
Could be purified based on
-size
-pI/charge
-etc.
Purify/separate fumarase
Manipulated gene
+~18 additional
nucleotides
Codes for fumarase
+ ~6 additional amino
acids at the C terminus
H-H-H-H-H-H
We’ve added a gene
to this strain of yeast!
Unique feature among proteins
Problem of protein stability
• “Proteolysis”
– Lysis of peptide bonds
• Typically catalyzed: proteases
– KEEP PROTEIN COLD
• Slow down proteolytic enzymes
– Chemical protease inhibitors
• Block enzyme action
Problem of protein stability
• Structural stability
– Native-like buffers
• Prevent drastic changes in pH, osmolarity
• Detergents can stabilize protein structure
– *Mild* eg. Igepal, Tween-20, etc.
– Many detergents destabilize protein structure,
eg. SDS
– Temperature
•
•
•
•
Room temp too warm?
Centrifugation
Generate lots of heat
Bead milling
Storage?
*Fumarase: relatively high affinity
for stationary phase
*Other proteins: mobile phase
*Weak fumarase’s affinity by
competition
INSOLUBLE “Bead”
H2O
COVALENT
http://tinyurl.com/ysn9ya
Company’s handbook (Qiagen)
H2O
• Fraction collector
• Pump
• Gradient maker
Anion exchange trial
• Start with three proteins
– Affinity for stationary vs. mobile phases
(partitioning)
• Change stationary phase/change partitioning
– Watch volume used for gradient
A: 25 mM Tris
“Gradient maker”
B: 25 mM Tris,
500 mM NaCl
Equal
volumes
B
A
To column
Start: 100% A
Volumes
decreased
but still equal
B
50%A
50%B
B >100%B
To column
Halfway through: 50% A
Column sees
0 mM NaCl
Column sees
250 mM NaCl
To column
Close to the end: almost 100% B
Column sees
~500 mM NaCl