Protein Analysis and Purification Analytical Separations Gel-electrophoresis IEF 2D-gels Preparative...
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Transcript of Protein Analysis and Purification Analytical Separations Gel-electrophoresis IEF 2D-gels Preparative...
Protein Analysis and Purification
Analytical SeparationsGel-electrophoresisIEF2D-gels
Preparative SeparationsVarious chromatographic methods
Total E. coli Proteins - 2-Dimensional Gel
Overview of Protein Purification
IntroductionProtein PropertiesTypes of SeparationsProtein Inactivation/Stabilization
Overexpression of Recombinant ProteinsPurification Tags/Affinity Handles
Protein PurificationObject: to separate a particular protein from all other proteins and cell components
There are many proteins (over 4300 genes in E. coli)
A given protein can be 0.001-20% of total protein
Other components:
nucleic acids, carbohydrates, lipids, small molecules
Enzymes are found in different states and locations:
soluble, insoluble, membrane bound, DNA bound,
in organelles, cytoplasmic, periplasmic, nuclear
Scale of Purification
Micro ng-g
Normal lab mg-100 mg
Large lab-scale gm
Pilot plant 10 g-kg
Industrial 100 tons
Most common in research lab
Amount & Purity Required for Different UsesUse Amount needed Purity Required
Immunology g-mg
Polyclonal antibodies High
Monoclonal antibodies Medium
Enzymology mg High (>95%)
Physical Properties mg-g High
Enzyme/protein Chemistry mg-g High
Structure g High
Research Enzymes g-mg Variable; must be free of key contaminants
Pharmaceutical g-kg Very high (>99.99%) free of bacterial endotoxin
Industrial Enzymes kg-ton Variable, often low
Study Question #1You are given a shoe box full of an assortment of small objects including:
Ping Pong balls
Sugar cubes
Paper clips
1/2” brass screws
Iron filings
1. List the properties of each of these components that might help you fractionate them.
2. Devise the most efficient method you can for getting pure paper clips.
20 Naturally-occurring Amino Acids
Acidic:
D, E, (C, Y)
Basic:
K, R, H
Hydrophobic:
I, L, V, W, F
Polar:
S, T, N, Q
Other:
G, A, M, P
Main Types of Molecular Interactions
Hydrogen Bonds
N H - - - - N N-H + N
low temperature high temperature
N H - - - - O C strength is very dependent on geometry donor acceptor and distance (2.6-3.1 A)
Hydrophobic Interactions (waxy residues: Ileu, Leu, Val, Phe, Trp)
high salt high temperature low salt
Ionic Interactions (charged residues:Asp- Glu- S- Lys+ Arg+ His+)
low ionic strength high ionic strength
-+ - +Cl- Na+
...
H2OH
HHH
H HHH
Variables that Affect Molecular Forces
Temperature
Ionic strength
Ion type
Polarity of solvent (dielectric constant)
pH
Protein Properties - Handles for FractionationSize (110 Da/amino acid residue)
smallest most proteins largest
Amino acids: 30 100 1,000 15,000
MW (kDa): 3.3 11 110 1,600
Multi-subunit complexes can contain 5-30 subunits
Shape
globular (sphere) asymmetric (cigar)
Effects frictional properties, effective radius, movement through poresCentrifuge Gel filtration
Sediments slower
Appears smaller
Elutes earlier
Appears larger
Protein Properties - Handles for FractionationNet chargeIonizable group pKa pH2 pH7 pH12C-terminal (COOH) 4.0 oooooooo----------------------------------------
Aspartate (COOH) 4.5 oooooooooo-------------------------------------
Glutamate (COOH) 4.6 ooooooooooo------------------------------------
Histidine (imidazole) 6.2 +++++++++++++oooooooooooooooooooo
N-terminal (amino) 7.3 +++++++++++++++oooooooooooooooooo
Cysteine (SH) 9.3 ooooooooooooooooooooooo-----------------
Tyrosine (phenol) 10.1 oooooooooooooooooooooooooo-------------
Lysine (amino) 10.4 ++++++++++++++++++++++++oooooooo
Arginine (guanido) 12.0 ++++++++++++++++++++++++++++++o
Isoelectric pointpI = pH where protein has zero net chargeTypical range of pI = 4-9
Charge distribution
++
++
-
--
-
uniform
++ +
+
-- - -
clusteredversus
Protein Properties-Handles for FractionationHydrophobicity Hydrophobic residues usually are buried internally
The number and distribution on the surface vary
Can use Hydrophobic Interaction Chromatography
Solubility Varies from barely soluble (<g/ml) to very soluble (>300 mg/ml)
Varies with pH, ionic strength/type, polarity of solvent, temperature
Least soluble at isoelectric point where there is least charge repulsion
Ligand and metal binding Affinity for cofactors, substrates, effector molecules, metals, DNA
When ligand is immobilized on a bead, you have an affinity bead
HHH
hydrophobic patch
Protein Properties-Handles for Fractionation
Reversible association e.g., E. coli RNA polymerase
Post-translational modifications Carbohydrates, lipids, phosphates, sulfates
Can be very useful purification handles
E.g.: Use of plant lectins to bind certain glycoproteins
Specific sequence or structure Precise geometric presentation of amino acids on surface of a protein
Epitope for binding to a specific antibody; use immunoaffinity column
Binding site for another protein; use protein affinity column
Monomer (0.3M NaCl) Dimer (0.05 M NaCl)
Separation Processes that can be Used to Fractionate Proteins
Separation Process Basis of SeparationPrecipitation ammonium sulfate solubility
polyethyleneimine (PEI) charge, size
isoelectric solubility, pI
Chromatography gel filtration (SEC) size, shape
ion exchange (IEX) charge, charge distribution
hydrophobic interaction(HIC) hydrophobicity
DNA affinity DNA binding site
immunoaffinity (IAC) specific epitope
chromatofocusing pI
Electrophoresis gel electrophoresis (PAGE) charge, size, shape
isoelectric focusing (IEF) pI
Centrifugation sucrose gradient size shape, density
Ultrafiltration ultrafiltration (UF) size, shape
Typical Protein Purification Scheme
Protein Inactivation/StabilizationBuffers Solution Components
Protein Sources for PurificationTraditional natural sources Bacteria, animal and plant tissue
Cloning recombinant proteins into overexpression vector/host systems for intracellular production (E. coli the most used)
In vitro protein synthesis Transcription/translation systems
Total E. coli Proteins - 2-Dimensional Gel
What You Can Learn from Amino Acid Sequence 1. Molecular weight of the polypeptide chain 2. Charge versus pH; Isoelectric point 3. Extinction coefficient 4. Hydrophobicity & membrane spanning regions 5. Potential modification sites 6. Conserved motifs that suggest cofactor affinity
What You Can’t Learn from Amino Acid Sequence
1. Function 2. 3-Dimensional structure; Shape 3. Multi-subunit features 4. Ammonium sulfate precipitation properties 5. Surface features (hydrophobic patches, charge
distribution, antigenic sites)
Conclusion: Protein Purification is still very empirical!
Engineering Proteins for Ease of Purification and Detection
Once you have a gene cloned and can over-express the protein, you can alter protein to improve the ease of purification or detection
You can fuse a tag to the N-or C- terminus of your protein
You can decide to remove the tag or not
Basic strategies
Add signal sequence that causes secretion into culture medium
Add protein that helps the protein refold and stay soluble
Add sequence that aids in precipitation
Add an affinity handle (by far the most used is the His-tag)
Add sequence that aids in detection
CSH Protein Course -Sigma32 Purification
MW A B C D E F G A/3 B/3 D/3
’
32
225
50
35
10 kDa