Biochemistry 412

Preparative Protein Chemistry

9 February 2007

The Three “Eras” of Protein Purification

1. The “Classical” (Pre-Recombinant DNA) Era (pre-1978)- Proteins purified from natural sources only

2. The Recombinant DNA (Pre-Genomic) Era (~1978 - late ‘90s)- Proteins purified from natural sources* and

recombinant cells

3. The Genomic and Post-Genomic Era(s) (late ‘90s - present)- Nearly all protein purification from recombinant cells,

since most information about proteins is now insequence (and other) databases

*Note: purification of proteins from natural sources was often motivated by theneed to get protein for amino acid sequencing so that oligonucleotide “probes”could be designed and used to clone the gene encoding the protein.

Purification schemes vary, depending on the source of the protein

and its intrinsic biophysical properties...

…some flow-charts for typical schemes follow.

Purification Scheme for Proteins from their Natural Source

Purification Scheme for Soluble Recombinant Proteins

Purification Scheme for Insoluble Recombinant Proteins

Purification Scheme for Membrane-Associated Proteins

Positively-charged basic residues (K, R, & H)

Negatively-charged acidic residues (E & D)

Hydrophobic “patch”

Ligand binding pocket(active site)

ca. 40 ÅMacromolecular

dimensions:

Proteins are Amphiphilic Macro-Ions

>>> The charged groups, hydrophobic regions, size, and solvation affect the biophysical properties of the protein and largely determine its purification behavior.

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ChromatographyLiquid flow

Liquid flow

4:37990909

Time 1 2 3 4 5

Separation according to: -molecular weight/ size-charge-hydrophobicity-affinity

Sample containing proteins or peptides

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Purity

Step

Capture

Intermediatepurification

Polishing

Isolate product,concentrate, stabilize

Remove bulkimpurities

Achieve final purity.Remove trace impurities,structural variants,aggregates, viruses, etc.

Three Phase Strategy: An aid in developing thepurification scheme

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Sample Preparation

General considerations:• Select extraction procedure according to source and

location of protein• Use gentle procedures to minimize acidification and

release of proteolytic enzymes• Work quickly at sub-ambient temperatures• Use buffer to maintain pH, ionic strength

Goal: To stabilize sample

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Always Limit the Number of StepsMaximize the Yield at Each Step

Number of steps

Yield (%)

95% / step

90% / step

85% / step80% / step75% / step

0

20

40

60

80

100

1 2 3 4 5 6 7 8

20% overallyield!

Gel Filtration

Gel Filtration (GF) Chromatography

The principle of gel filtration -- excluded volume[Note: gel filtration chromatography is also sometimes

called “size exclusion chromatography”]

Vo = “void volume”Vt = “bed volume”Ve = “elution volume”Vi = Vt - Vo

Principles of gel chromatography (con’d)

Gel Filtration Elution Volumes as a Function of Molecular Weight

Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.

Ion Exchange Chromatography

Ion Exchange (IEX) Chromatography

Ion Exchange Chromatography (con’d)

Cation exchangecolumn

Anion exchangecolumn

Some other popular chromatographic methods:

• Hydrophobic interaction chromatography

• Affinity chromatography

• Reverse phase chromatography

Hydrophobic Interaction Chromatography (HIC)

Affinity Chromatography

“Reversed Phase” Chromatography (RPC)(elution with organic solvents)

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Technique End conditionsStart conditions

Small sample volume GF Diluted sampleBuffer change (if required)

Low ionic strength IEX High ionic strength orpH change

High ionic strength HIC Low ionic strength

Specific binding conditions AC Specific elution conditions

Linking Chromatography Techniques

Note: after IEX, HIC, or AC, sample isconcentrated, too.

It is good to design your purification to have the start conditions ofeach step match the end conditions of the previous step in order toavoid intervening buffer exchange steps, which add to your losses.

In addition, there are non-chromatographicprotein purification techniques, e. g.:

• Ammonium sulfate precipitation

• Sedimentation (rare)

• Recombinant gene product over-expression

• Inclusion body prep (see earlier slide)

• Detergent extraction

• Heat treatment (especially for recombinant thermophile proteins expressed in E. coli)

• Etc.