Biochemistry 4122004

20 February Lecture

Analytical & Preparative Protein Chemistry II

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

Negatively-charged acidic residues (E & D)

Hydrophobic “patch”

Ligand binding pocket(active site)

ca. 40 Å

Macromoleculardimensions:

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.

5

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 the purification 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 Steps

Maximize the Yield at Each Step

Number of steps

Yield (%)

95% / step

90% / step

85% / step

80% / 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)

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

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

• Ammonium sulfate precipitation

• Sedimentation (rare)

• Recombinant gene product over-expression

• Refractile body prep (see above)

• Detergent extraction

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

• Etc.

Once You’ve Purified Your Protein,How Do You Characterize It?

Some typical analytical tests:

- SDS PAGE (both reducing and non-reducing)- Bioassay (if you have one)- Total protein determination- UV spectrophotometry- CD spectrometry- disulfides?- amino acid analysis- N-terminal (& C-terminal?) sequencing- HPLC?- metal analysis- mass spectrometry- NMR spectrometry & X-ray crystallography*- other?

*Not usually used for routine analytical purposes!!

Protein Size Determination by SDS Polyacrylamide Gel Electrophoresis

Adapted fromT. E. Creighton,

ProteinsW.H.Freeman,

1984+ electrode

- electrode

Circular Dichroism Spectroscopy of Polypeptides

Adapted fromT. E. Creighton, ProteinsW.H.Freeman,1984