Biochemistry 412

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Biochemistry 412 2004 20 February
Lecture Analytical Preparative Protein
Chemistry II
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Proteins are Amphiphilic Macro-Ions
Positively-charged basic residues (K, R, H)
Hydrophobic patch
Macromolecular dimensions
ca. 40 Å
Ligand binding pocket (active site)
Negatively-charged acidic residues (E D)
gtgtgt 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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Chromatography
Sample containing proteins or peptides
Liquid flow
Liquid flow
Separation according to -molecular weight/
size -charge -hydrophobicity -affinity
Time
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437 990909
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Three Phase Strategy An aid in developing the
purification scheme
Achieve final purity. Remove trace impurities,
structural variants, aggregates, viruses, etc.
Purity
Polishing
Remove bulk impurities
Intermediate purification
Isolate product, concentrate, stabilize
Capture
Step
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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
Yield ()
100
80
95 / step
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90 / step
40
85 / step
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20 overall yield!
80 / step
75 / step
0
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Number of steps
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Gel Filtration
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Gel Filtration (GF) Chromatography
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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
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Principles of gel chromatography (cond)
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Gel Filtration Elution Volumes as a Function of
Molecular Weight
Adapted from T. E. Creighton, Proteins,
W.H.Freeman,1984.
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Ion Exchange Chromatography
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Ion Exchange (IEX) Chromatography
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Ion Exchange Chromatography (cond)
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Some other popular chromatographic methods
Hydrophobic interaction chromatography
Affinity chromatography Reverse phase
chromatography
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Hydrophobic Interaction Chromatography (HIC)
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Affinity Chromatography
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Reversed Phase Chromatography (RPC) (elution
with organic solvents)
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Linking Chromatography Techniques
Technique
End conditions
Start conditions
Small sample volume
GF
Diluted sample Buffer change (if required)
IEX
Low ionic strength
High ionic strength orpH change
High ionic strength
HIC
Low ionic strength
Specific binding conditions
AC
Specific elution conditions
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In addition, there are non-chromatographic protein
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.
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Once Youve 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!!
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Protein Size Determination by SDS Polyacrylamide
Gel Electrophoresis
- electrode
Adapted from T. E. Creighton, Proteins W.H.Freema
n, 1984
electrode
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Circular Dichroism Spectroscopy of Polypeptides
Adapted from T. E. Creighton, Proteins W.H.Freema
n, 1984
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