Protein Purification Strategies
Transcript of Protein Purification Strategies
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Protein Purification
Tutorial
Minnie MurugesanDr. Scotts Group
07-12-05
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Eventful Protein Purification
Grow cells in media (vector+tag) Centrifuge, Collect the pellet
Lyse the cells (appropriate buffer)
Purification Strategy
Pilot ExpressionSDS PAGE, Assay
Solubility
Aggregation
Recombination
Characterization
Mass Spectroscopy
X-rayCrystallography
Functional Assay
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OUTLINE
Chromatography techniques AffinityChromatography (AC)
Hydrophobic Interaction Chromatography (HIC)
Ion Exchange Chromatography (IEC)
Gel Filtration (GF)
Capillary electrochromatography (CEC)
Other New Strategies for Protein Purification
Solubility, Aggregation and Re-folding of Proteins
Invented by a Russian botanist named Mikhail Tswett in 1903. He separated
plant pigments using glass columns packed with calcium carbonate.
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1. Evaluate an assay for the protein of interest
2. Shortlist a method to have a reasonable source for that activity
Protein Purification Strategies
(http://www5.amershambiosciences.com/aptrix/upp00919.nsf/Content/LabSep_EduC%5CAboutPurBiom%5CHowToCombine)
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Three Phase Strategy
For Membrane Proteins
(www.amershambiosciences.com)
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Chromatographic Mode Acronym Separation Principle
Non-interactive modes of liquid chromatography
Size-exclusion chromatography SEC Differences in molecular size
Slalom chromatography (forDNA) - Diff. in length and flexibility
Interactive modes of liquid chromatography
Ion-exchange chromatography IEC Electrostatic interactions
Normal-phase chromatography NPC Polar interactions
Reversed-phase chromtography RPC Dispersive interactions
Hydrophobic interactionchromatography
HIC Dispersive interactions
Affinity chromatography AC Biospecific interaction
Metal interaction chromatography MIC Complex w/ an immobilized metal
Chromatographic Modes of Protein Purification
(Christian G.Huber, BiopolymerChromatography,Encylcopedia in analytical chemistry, 2000)
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AffinityChromatography
Surface bound with
Epoxy, aldehyde or aryl ester groups
Metal Interaction Chromatography
Surface bound with
Iminodiacetic acid + Ni2+/Zn2+/Co2+
Affinity Chromatography
(Christian G.Huber, BiopolymerChromatography,Encylcopedia in analytical chemistry, 2000)
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Metal Interaction Chromatography (AC)
Points to Note:1. Avoid chelating agents
2. Increasing incubation time
3. Slow gradient elution
(www.qiagen.com)
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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
~15Qmol Ni2+
Backpressure ~43psi
Change the guard column filter
(Christian G.Huber, BiopolymerChromatography,Encylcopedia in analytical chemistry, 2000)
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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.
Temperatureplays 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
(Christian G.Huber, BiopolymerChromatography,Encylcopedia in analytical chemistry, 2000)
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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 whileinteracting with tentacle ion exchanger
(www.novagen.com)
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Gel Filtration
(http://lsvl.la.asu.edu/resources/mamajis/chromatography/chromatography.html)
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Immuno Affinity Chromatography
(http://www.cellmigration.org/resource/discovery/discovery_proteomics_approaches.html)
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ATP immobilized on polyacrylamide resin
DNA Binding Proteins
Heparin Sepharose
Negatively charged proteins (pI >7) are not captured/separated effectively.
(www.novagen.com)
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Capillary Electrochromatography
CEC is an electrokinetic separation technique
Fused-silica capillaries packed with stationary phase
Separation based on electroosmotically 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/N
number of plates N = 16(t/W)2
whereL = column length, t = retention time, and W = peak width at baseline
(http://www.capital-hplc.co.uk)
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GSTBind Purification KitsHisBind Purification Kits
Magnetight Oligo d(T) Beads
MagPrep Streptavidin Beads
Protein A and Protein G Plus Agaroses
STag Purification KitsStreptavidin Agarose
T7Tag 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 BiotekCorporation)
ProteoSpinDetergent 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
(http://www.emdbiosciences.com)
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Schematic of a Multi-dimensional Separation System
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Fast Protein Liquid Chromatograph (FPLC)
1
2
3
5
4
No air bubbles
(Priming) Use degassed buffers
Injector Module
Column Inlet
Detector
Fraction
Collector
(www.pharmacia.com)
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Protein Analysis
(http://www.cellmigration.org/resource/discovery/discovery_proteomics_approaches.html)
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Reagent Derivatization Detection
o-PhthaldialdeyhdePrecolumn/
PostcolumnFL, Ex 340nm/Em 400nm
Fluorescamine Precolumn/Postcolumn
FL, Ex 390nm/Em 490nm
Indocyanine greeen PrecolumnFL, Ex 765nm/
Em 820-840nm
Detection of Proteins byDerivatization with Higher Sensitivity
1000 times more sensitive than UV-Vis detection
(Christian G.Huber,Encylcopedia in analytical chemistry, 2000)
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Solubility of a protein
Membrane proteins
1. 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).
(http://www.ls.huji.ac.il/~purification)
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Protein Aggregation
(http://www.integritybio.com/protein%20aggregation.htm)
Numerous physicochemical stresses can induce protein aggregation:
Heat, pressure, pH, agitation, freeze-thawing, dehydration, heavy metals,
phenolic compounds, and denaturants.
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Solubilization of Aggregated Proteins
Denaturation and Renaturation
Variables Good starting point
Buffer composition (pH, ionic strength) 50 mM Tris-HCl, pH 7.5
Incubation temperature 30C
Incubation time 60 min
Concentration of solubilzing 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 allowformation 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.
(www.biovectra.com)
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Polyethylene glycol
(PEG 3350)
0.1-0.4 g/L L-Arginine hydrochloride 0.4-0.8M
Nondenaturating
concentrations ofUrea
< 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 ~1M
Methylfomamide 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
(http://www.ls.huji.ac.il/~purification)
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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
Na2SO
4or K
2SO
40.4-0.6 M Dodecyl Maltoside 2.0-5.0 mM
Cyclodextrin 20-100 mM Sarkosyl 0.05-0.5 %
Reagents used for Re-folding of proteins (Continued)
(http://www.ls.huji.ac.il/~purification)
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6xHis Tagged Protein Detection Directly on the Gel (from Pierce)
E. colilysates expressing 6xHis-tagged proteins,stained withthe Pierce
6xHis Protein Tag Staining Kit
(www.piercenet.com)
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(www.biotech.ou.edu)
University ofOklahoma
Recombinant Protein Solubility Prediction
Type (or cut and paste) your protein sequence below, click on the "Submit" button, and
the solubility probability ofyour protein will be calculated.
The statistical model predicts protein solubilityassuming the protein is being over-expressed in Escherichia coli.
The input protein sequence has a 73.4 percent chance ofinsolubility when
overexpressed in E. coli. - mbh8 NADH dehydrogenase subunit - integral
membrane protein (NP_579159)The input protein sequence has a 80.5 percent chance ofsolubility
when overexpressed in E. coli. - replication factor A related protein (NP_579749)