protein purification
DESCRIPTION
Protein PurificationTRANSCRIPT
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PRESENTATION OUTLINE•Protein Purification Scheme•Protein fractionation •Chromatography techniques
• Affinity Chromatography (AC)• Hydrophobic Interaction Chromatography (HIC)• Ion Exchange Chromatography (IEC)• Gel Filtration (GF)• Capillary electrochromatography (CEC)
• Strategies for Protein Purification Solubility, Aggregation and Re-folding of Proteins
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Sample Separationtechnique
Fractionation
Purification is a Multi-Step Procedure.
Is there activity?Set aside No
CombineFractionsyes Monitor purity
Assay total protein
Assay enzyme activity
Pure?
Prepare for analytical technique
yes
No
Repeat with another
separationtechnique until
pure
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General Protein Purification Scheme
• Grow cells in media (vector+tag)•Bacteria Suspension•Bioreactor
Purification Strategy
ExpressionSDS PAGE Assay
SolubilityAggregation
Recombination
CharacterizationMass Spectroscopy
X-ray CrystallographyFunctional Assay
Lyse the cells (appropriate buffer)Centrifuge Collect the pellet
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1. Evaluate an assay for the protein of interest2. Shortlist a method to have a reasonable source for that activity
Set Protein Purification Strategy
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Preparing the sample (Crude Extract)
Protein from cells or tissue
Microbial cells or tissue
Break cells,
Blender, homogenizer, sonication,pressureosmotic Pellet with intact
cells, organelles, membranes and membrane proteins
Supernatant withSoluble protein
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• As the column separates the proteins in the mixture, the “effluent” drips into a series of fraction tubes that are moving at a specific rate of speed. These tubes are called fractions.
• Here we are showing 20 tubes. Fraction collectors in most labs have about 75-200 tubes.
• How do we know which fractions contain protein? Total protein a can be estimated by taking the absorbance at 280 nm in a spectrophotometer. Aromatic amino acids absorb light at this wavelength causing all proteins to have absorbance at 280nm. Many fraction collectors measure the A280 as the column is running.
Collect fractions.
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A280
Plot values
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction #
Question 1. How do we know which fractions contain protein?
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• Total protein a can be estimated by taking the absorbance at 280 nm in a spectrophotometer.
• The values can be plotted against the fraction number in is what is called an elution profile.
• Notice the peaks on the graph. These indicate where the fractions are that contain protein.
Question 1. How do we know which fractions contain protein?
A2800 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Fraction#
A280
Fraction #
Peaks
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• Enzyme activity can be determined by performing an enzyme assay on each fraction that contains protein.
Which fractions contained the desired protein?
A2800 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction#
A280
Fraction #
Enz. Assay.
Fraction#
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• Enzyme activity can be determined by performing an enzyme assay on each fraction that contains protein.
• Notice the results of the enzyme assay. The highest activity corresponds to one of the peaks.
• Now we can have them discard tubes that don’t have enzyme activity.
Which fractions contained the desired enzyme?
A2800 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction#
A280
Fraction #
EnzAssayResults
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The Way to Chromatography
• In order to isolate sufficient quantities of protein, you may need to start with kilogram quantities of source (i.e. bacteria, tissues, etc.) These amounts can best be handled using precipitation methods (e.g. ammonium sulfate precipitation). Later in the purification, large columns can be used to handle gram to milligram quantities. Amounts handled on gels are typically in microgram quantities.
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Property Methods
Solubility Precipitation with ammonium sulfate (salting out)*
Size / shape Size-exclusion chromotography
Isoelectricpoint (charge)
Ion exhange chromatography
binding to small molecules
Affinity chromatography
Common methods of protein purification
*Ammonium sulfate precipitation is cheap, easy, and accommodates large sample sizes. It is commonly one of the first steps in a purification scheme.
• Purification procedures attempt to maintain the protein in native form. Although some proteins can be re-natured, most cannot!
• To purify a protein from a mixture, biochemists exploit the ways that individual proteins differ from one another. They differ in:
• Thermal stability: For most protein purifications, all steps are carried out at ~5°C to slow down degradation processes.
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Picture of protein gel with lanes showing sequential purification steps
Procedure Fraction vol(ml)
Total Prot(mg)
Activity(units)
Specific activity
Units/mg
Crude cellular extract
1400 10000 100,000 10
Size-exclusion
90 400 80,000 200
Ion exchange
80 100 60,000 600
Note: The type and order of steps are customized for each protein to be purified. An effective purification step results in a high yield (minimal loss of enzyme activity) and large purification factor (large increase in specific activity).
Purification Yield
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Chromatographic Mode Acronym Separation Principle
Non-interactive modes of liquid chromatography
Size-exclusion chromatography SEC Differences in molecular size
Agarose chromatography (for DNA) for DNA binding proteins
- Diff. in length and flexibility
Interactive modes of liquid chromatographyIon-exchange chromatography IEC Electrostatic interactions
Normal-phase chromatography NPC Polar interactions
Reversed-phase chromtography RPC Dispersive interactions*
Hydrophobic interaction chromatography HIC Dispersive interactions*
Affinity chromatography AC Biospecific interaction
Metal interaction chromatography MIC Complex w/ an immobilized
metal
Chromatographic Modes of Protein Purification
* Induced dipole – induced dipole interactions
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Column Selection
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Affinity ChromatographySurface bound with
Epoxy, aldehyde or aryl ester groups
Metal Interaction Chromatography
Surface bound withIminodiacetic acid +
Ni2+/Zn2+/Co2+
Affinity Chromatography
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Metal Interaction Chromatography (AC)
Points to Note:
1. Avoid chelating agents
2. Increasing incubation time
3. Slow gradient elution
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Affinity Chromatography
Binding Capacity (mg/ml) medium 12mg of histag proteins (MW= 27kDa)Depends on Molecular weight
Degree of substitution /ml medium~15mmol Ni2+
Backpressure ~43psiChange the guard column filter
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Biopolymer (phenyl agarose - Binding Surface)
Driving force for hydrophobic adsorptionWater molecules surround the analyte and the binding surface.
When a hydrophobic region of a biopolymer binds to the surface of a mildly hydrophobic stationary phase, hydrophilic water molecules are effectively released from the surrounding hydrophobic areas causing a thermodynamically favorable change in entropy.
Temperature plays a strong role
Ammonium sulfate, by virtue of its good salting-out properties and high solubility in water is used as an eluting buffer
Hydrophobic Interaction Chromatography
Hydrophobic region
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ION –EXCHANGE 1 • First, to determine the
charge on a protein, given its pI and the pH.
• Ion-exchange column chromatography separates proteins on the basis of charge.
• We will start with 4 proteins.
• pH 7.2• Positive charged column
60 Kd
Low pI (6)
20 Kd
Low pI (7)
20 Kd
Medium pI (7)
5 Kd
Hi pI (8)
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pos
• The matrix of an ion exchange is positively charged.
• What do you think will happen?
pos
pos
pos
pos
pos
pos
Run columnpos
pos pos
pos
pos
pos
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• The matrix of an ion exchange is positively charged.
• Only the pos charged proteins run through the pos charged column. The others “stick” to the column.
pos
pos
pos
pos
pos
pos
pos
pos
pos pos
pos
pos
pos
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Fractogel matrix is a methacrylate resin upon which polyelectrolyte Chains (or tentacles) have been grafted. (Novagen)
Ion Exchange Chromatography
Globular Protein
Deformation due to interaction with conventional ion
exchanger
Maintenance of conformation while
interacting with tentacle ion exchanger
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Gel Filtration
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• Gel filtration column chromatography separates proteins on the basis of size.
• We will start with 4 proteins.• You will want to purify the
“yellow one” 60 Kd
Low pI (6)
20 Kd
Low pI (7)
20 Kd
Medium pI (7)
5 Kd
Hi pI (8)
Gel Filtration
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• The matrix of a size-exclusion chromatography column is porous beads.
Run column
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• The matrix of a gel filtration column are beads with pores.
• The large gray proteins can’t fit in pores so flows faster.
• The red / yellow medium sized proteins get trapped in the pores.
• The black small proteins stay trapped in pores longer.
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Immune Affinity Chromatography
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ATP immobilized on polyacrylamide resin
DNA Binding Proteins
Heparin SepharoseNegatively charged proteins (pI >7) are not captured/separated effectively.
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Capillary Electrochromatography• CEC is an electrokinetic separation technique• Fused-silica capillaries packed with stationary phase • Separation based on electro-osmotically driven flow• Higher selectivity due to the combination of chromatography and electrophoresis
Fused silica tube filled with porous methacrylamide-stearyl methacrylate-dimethyldiallyl ammonium chloride monolithic polymers, 80 x 0.5mm i.d., 5.5kV. High Plate count ~ 400,000
Height Equivalent to a Theoretical Plate /Plate Count (HETP) H = L/Nnumber of plates N = 16(t/W)2 where L = column length, t = retention time, and W = peak width at baseline
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CEC columns AC, IEC columns
CEC columnNP, RP columns
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Schematic of a Multi-dimensional Separation System
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
Fast Protein Liquid Chromatograph (FPLC)
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3
5
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• No air bubbles (Priming)• Use degassed buffers
Injector Module
Column Inlet
DetectorFractionCollector
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Protein Analysis
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Reagent Derivatization Detection
o-Phthaldialdeyhde
Precolumn/ Postcolumn FL, Ex 340nm/Em 400nm
Fluorescamine Precolumn/ Postcolumn FL, Ex 390nm/Em 490nm
Indocyanine greeen Precolumn FL, Ex 765nm/
Em 820-840nm
Detection of Proteins by Derivatization with Higher Sensitivity
1000 times more sensitive than UV-Vis detection
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Solubility of a Protein
Membrane proteins1. Removal of unbroken cells from the cell lysate by low speed
centrifugation (20 min at 10,000 g). 2. Isolation of the membrane particles from the supernatant by
ultracentrifugation (60 min at >100 000 g). 3. Washing of the membrane particle to remove all soluble proteins. 4. Solubilization of protein from the membrane particles by a mild
detergent. (detergent: protein ratio = 1:10)5. Phosphate buffers(0.1M-0.5M), 5-50% glycerol helps.
• Depends strongly on the composition of the lysis buffer.• Salt concentration
Freeze-thaw protocol* Freeze quickly on dry ice and leave for 3 min. * Thaw immediately at 42 °C. Vortex vigorously to mix well. * Repeat the two previous steps three more times (4 cycles in all).
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Protein Aggregation
• Numerous physicochemical stresses can induce protein aggregation:
• Heat, pressure, pH, agitation, freeze-thawing, dehydration, heavy metals, phenolic compounds, and denaturants.
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Denaturation and Renaturation
Variables Good starting pointBuffer composition (pH, ionic strength) 50 mM Tris-HCl, pH 7.5Incubation temperature 30°CIncubation time 60 minConcentration of solubilizing agent 6 M guanidine-HCl or 8 M ureaTotal protein concentration 1-2 mg/ml
Re-folding of Proteins
The addition of a mixture of reduced and oxidized forms of low molecular weight thiol reagent usually provides the appropriate redox potential to allow formation and reshuffling of disulfide bonds (1-3 mM reduced thiol and a 5:1 to 1:1 ratio of reduced to oxidixed thiol)
The most commonly used are glutathione, cysteine and cysteamine.
Solubilization of Aggregated Proteins
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Polyethylene glycol (PEG 3350)
0.1-0.4 g/L L-Arginine hydrochloride
0.4-0.8M
Nondenaturating concentrations of Urea
< 2.0 M K-Glutamate ~5M
Nondenaturating concentrations Gdm/ClH
< 1.0 M Proline ~1M
Methylurea 1.5-2.5 M Glycerol 20-40 %Ethylurea 1.0-2.0 M Sorbitol 20-30 %Formamide 2.5-4.0 M Sucrose ~1MMethylfomamide 2.0-4.0 M Trehalose ~1M
Acetamide 1.5-2.5 M TMAO (trimethylamine N-oxide)
~1M
Ethanol Up to 25% Sulfo Betaine ~1M
Reagents used for Re-folding of proteins
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n-Penthanol 1.0-10.0 mM Lauryl Maltoside 0.06 mg/ml
n-Hexanol 0.1-10.0 mM CETAB 0.6 mg/ml
Cyclohexanol 0.01-10.0 mM
CHAPS 10-60 mM
Tris
> 0.4 M Triton X-100 10 mM
Na2SO4 or K2SO4 0.4-0.6 M Dodecyl Maltoside
2.0-5.0 mM
Cyclodextrin 20-100 mM Sarkosyl 0.05-0.5 %
Reagents used for Re-folding of proteinsCont.
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6xHis Tagged Protein Detection Directly on the Gel (from Pierce)
E. coli lysates expressing 6xHis-tagged proteins, stained with the Pierce 6xHis Protein Tag Staining Kit
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GST•Bind™ Purification KitsHis•Bind® Purification KitsMagnetight™ Oligo d(T) BeadsMagPrep® Streptavidin BeadsProtein A and Protein G Plus AgarosesS•Tag™ Purification KitsStreptavidin AgaroseT7•Tag™ Affinity Purification KitProteoSpin™ CBED (Concentration, Buffer Exchange and Desalting) Maxi Kit — Effectively desalts and concentrates up to 8 mg of protein with an efficient, easy-to-use protocol.(Norgen Biotek Corporation)ProteoSpin™ Detergent Clean-up Micro Kit — Provides a fast and effective procedure to remove detergents including SDS, Triton® X-100, CHAPS, NP-40 and Tween 20.
Commercially available protein purification kits
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REFERENCES• Christian G. Huber, Biopolymer Chromatography,
Encylcopedia in analytical chemistry, 2000• www.qiagen.com• www.novagen.com• http://lsvl.la.asu.edu/resources/mamajis/
chromatography/chromatography.html • http://www.cellmigration.org/resource/discovery/
discovery_proteomics_approaches.html • http://www.capital-hplc.co.uk• http://www.ls.huji.ac.il/~purification• www.biovectra.com• http://www.ls.huji.ac.il/~purification