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.
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!
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
Gel Filtration Elution Volumes as a Function of Molecular Weight
Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.
Some other popular chromatographic methods:
• Hydrophobic interaction chromatography
• Affinity chromatography
• Reverse phase chromatography
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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.