Protein Purification

Why purify proteins?

1. Biochemically reconstitute a
cellular process - can define the
minimal components required and
learn about the mechanism
2. Identify the molecular basis of an
activity
3. Drug development
4. Generate antibodies
5. Fishing expeditions (probe to
identify interactors)
Reconstitute an activity with
purified components

• Develop an assay

• Use conventional chromatography to purify

the components responsible for the activity

• Example: Farnesyltransferase
Cholesterol
Biosynthetic
Pathway
Reaction Catalyzed by Farnesyltransferase

Ras + FPP Farnesyl-Ras + PPi

What do you need to set up
an assay for FTase activity?
Assay: Mix components and incubate. Stop reaction by
precipitation. Collect pptd. protein on filter and measure
radioactivity incorporated into p21H-ras.
 How to “measure”purification?

• Purification based on activity

• Quantitate activity
• Measure total protein
• Determine specific activity = U/mg
• Fold purification is ratio of specific activities
Purification Table
 Step 1: Ammonium sulfate
fractionation

• Homogenize 50 rat brains

• 60,000 x g centrifugation
• 30% ammonium sulfate cut of
supernatant
• Dissolve precipitate in buffer
• Dialysis
 Distribution of Charge and Hydrophobic
Patches over Surface of a Typical Protein

SOLUBILITY
• Polar interactions with aqueous solvents
• Ionic interactions with salts
• Repulsive interactions between charged
molecules

From: Protein Purification: Principles and Practice (R.K. Scopes)

 Proteins are Least Soluble Near their
Isoelectric Point

• Metal ‘salt’ of a protein

generally has a higher
isoelectric point.

From: Protein Purification: Principles and Practice (R.K. Scopes)

 • Dependent on hydrophobicity of
Salting out protein
• Salt ions become solvated, pulling
ordered water away from
hydrophobic patches
• More hydrophobic proteins
precipitate sooner

Strategy
• Encourage hydration of polar
regions
• Dehydration of hydrophobic regions
• No direct interaction with proteins
• In high salt, the solubility of
proteins generally decreases with
increasing temperature. Why?

Na Chaotropic salts that

K salts (not so soluble) destabilize structure
Pure enzyme
NH4 (can’t use pH>8, Why?) Potassium thocyanate
Mixture (3M)
(coaggregation) Sulfates Potassium iodide (2M)
Citrates (can’t use <pH 7) MgCl2 (4M)
phosphates Urea (8M) (denaturant)
GuHCl (6M) (denaturant)

From: Protein Purification: Principles and Practice (R.K. Scopes)

Step 2: Ion Exchange
 Ion Exchange Chromatography

From: Protein Purification: Principles and Practice (R.K. Scopes)

 Ion Exchange
Chromatography

from Freifelder Table 5.4. Choice of an Ion Exchanger for Purification of a Protein with a Known
Isolectric Point (Assuming that the protein is stable only in the pH range 5.5 – 8.5)
Isoelectric Point Ion exchange Buffer pH
8.5 Cation < or = 7.0
7.0 Cation < or = 6.0
Anion > or = 8.0
5.5 Anion > or = 6.5

From: Protein Purification: Principles and Practice (R.K. Scopes)

 Step 3: Affinity Chromatography
6 amino acid peptide: C-terminal amino acids of K-Ras
SDS-PAGE
Purification Table
Step 4: Gel Filtration
 Separation by size (and shape):
gel filtration chromatography

Stokes radius: hydrodynamic radius - contributions by mass, shape, and bound solvent.
A more extended molecule will have a larger Stoke's radius compared to a more compact
molecule of the same molecular weight.
Purified FTase is a Heterodimer

How do you go from

purified protein to the
genes that encode
FTase
and ?
 Farnesyltransferase
Structure

Crystal Structure of Protein Farnesyltransferase at 2.25 Angstrom Resolution

Hee-Won Park, Sobha R. Boduluri, John F. Moomaw, Patrick J. Casey, Lorena S. Beese *
Science (1997)275:1800-1805