Protein Purification

Why purify proteins?

1. Biochemically reconstitute acellular process - can define theminimal components required andlearn about the mechanism

2. Identify the molecular basis of anactivity

3. Drug development4. Generate antibodies5. Fishing expeditions (probe to

identify interactors)

Reconstitute an activity withpurified components

• Develop an assay

• Use conventional chromatography to purifythe components responsible for the activity

• Example: Farnesyltransferase

Cholesterol BiosyntheticPathway

Ras + FPP Farnesyl-Ras + PPi

Reaction Catalyzed by Farnesyltransferase

What do you need to set upan assay for FTase activity?

Assay: Mix components and incubate. Stop reaction byprecipitation. Collect pptd. protein on filter and measureradioactivity 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 sulfatefractionation

• Homogenize 50 rat brains• 60,000 x g centrifugation• 30% ammonium sulfate cut of

supernatant• Dissolve precipitate in buffer• Dialysis

Distribution of Charge and HydrophobicPatches over Surface of a Typical Protein

SOLUBILITY• PPolar interactions with aqueous solvents• IIonic interactions with salts• RRepulsive interactions between chargedmolecules

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

Proteins are Least Soluble Near theirProteins are Least Soluble Near theirIsoelectric PointIsoelectric Point

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

• Metal ‘salt’ of a proteingenerally has a higherisoelectric point.

Salting out

Pure enzyme

Mixture(coaggregation)

• Dependent on hydrophobicity ofprotein

• Salt ions become solvated, pullingordered water away fromhydrophobic patches

• More hydrophobic proteinsprecipitate sooner

Strategy

• Encourage hydration of polarregions

• Dehydration of hydrophobic regions

• No direct interaction with proteins

• In high salt, the solubility ofproteins generally decreases withincreasing temperature. Why?

NaK salts (not so soluble)NH4 (can’t use pH>8, Why?)

SulfatesCitrates (can’t use <pH 7)phosphates

Chaotropic salts thatdestabilize structurePotassium thocyanate(3M)Potassium iodide (2M)MgCl2 (4M)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)

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 Freifelder

Ion Exchange

Chromatography

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

Step 3: Affinity Chromatography6 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 ?

FarnesyltransferaseStructure

Crystal Structure of Protein Farnesyltransferase at 2.25 Angstrom ResolutionHee-Won Park, Sobha R. Boduluri, John F. Moomaw, Patrick J. Casey, Lorena S. Beese *Science (1997)275:1800-1805