Biochemistry 412 2004 20 February Lecture Analytical & Preparative Protein Chemistry II.
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Transcript of Biochemistry 412 2004 20 February Lecture Analytical & Preparative Protein Chemistry II.
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Biochemistry 4122004
20 February Lecture
Analytical & Preparative Protein Chemistry II
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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.
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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 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!
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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 (con’d)
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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 (con’d)
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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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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
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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.
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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!!
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Protein Size Determination by SDS Polyacrylamide Gel Electrophoresis
Adapted fromT. E. Creighton,
ProteinsW.H.Freeman,
1984+ electrode
- electrode
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Circular Dichroism Spectroscopy of Polypeptides
Adapted fromT. E. Creighton, ProteinsW.H.Freeman,1984
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