Transcript Current Approaches to Protein Purification Richard Burgess

Protein Purification
(from a lecture by Dr. Richard Burgess, University of Wisconsin, Madison, at the CSH protein
purification course).
Object: 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
Study Question
You 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 Naturallyoccurring 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
Overview of Protein Purification
Types of Separations
Protein Properties
Protein Inactivation/Stabilization
Protein Analysis and Purification
Analytical Separations
Gel-electrophoresis
IEF
2D-gels
Preparative Separations
Various chromatographic methods
Total E. coli Proteins - 2-Dimensional Gel
Main Types of Molecular Interactions
Hydrogen Bonds
N H----N
N-H +
low temperature
high temperature
N H----O C
donor
acceptor
N
strength is very dependent on geometry
and distance (2.6-3.1 A)
Hydrophobic Interactions (waxy residues: Ileu, Leu, Val, Phe, Trp)
HH
HH
H
H
H
H
H2O
high salt
high temperature
low salt
Ionic Interactions (charged residues:Asp- Glu- S- Lys+ Arg+ His+)
+... low ionic strength
Cl-
+
Na+
-
high ionic strength
Variables that Affect Molecular Forces
Temperature
Ionic strength
Ion type
Polarity of solvent (dielectric constant)
pH
Protein Properties - Handles for Fractionation
Size (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 pores
Centrifuge
Gel filtration
Sediments slower
Elutes earlier
Appears smaller
Appears larger
Protein Properties - Handles for Fractionation
Net charge
Ionizable group
pKa
pH2
pH7
pH12
C-terminal (COOH)
Aspartate (COOH)
Glutamate (COOH)
Histidine (imidazole)
N-terminal (amino)
Cysteine (SH)
Tyrosine (phenol)
Lysine (amino)
Arginine (guanido)
4.0
4.5
4.6
6.2
7.3
9.3
10.1
10.4
12.0
oooooooo---------------------------------------oooooooooo------------------------------------ooooooooooo-----------------------------------+++++++++++++oooooooooooooooooooo
+++++++++++++++oooooooooooooooooo
ooooooooooooooooooooooo----------------oooooooooooooooooooooooooo------------++++++++++++++++++++++++oooooooo
++++++++++++++++++++++++++++++o
Isoelectric point
pI = pH where protein has zero net charge
Typical range of pI = 4-9
Charge distribution
+
-
+
-
uniform
-
+
-
+
+
versus
+
+
+
clustered
-
-
-
-
Protein Properties-Handles for Fractionation
Hydrophobicity
Hydrophobic residues usually are buried internally
The number and distribution on the surface vary
Can use Hydrophobic Interaction Chromatography
H HH
hydrophobic patch
Solubility
Varies from barely soluble (<mg/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
Separation Processes that can be Used
to Fractionate Proteins
Separation Process
Basis of Separation
Precipitation
ammonium sulfate
polyethyleneimine (PEI)
isoelectric
solubility
charge, size
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
Ultrafiltration
sucrose gradient
size shape, density
ultrafiltration (UF)
size, shape
Typical Protein Purification Scheme
Protein Inactivation/Stabilization
Buffers Solution Components
Protein Sources for Purification
Traditional 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
Determining the protein sequence from gel
(proteomics)
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
225
bb’
50
35
10 kDa
s32