•
The challenge of protein purification becomes self-evident when one
considers the complex mixture of macromolecules present in a biological
matrix such as a cell or tissue extract.
• Several thousand other proteins with different properties are present in any
given cell type (~5,000–8,000 proteins)
• Nonproteinaceous materials
• DNA
• RNA
• Polysaccharides
• Lipids
Actin – 1,000,000
molecules per cell
Transcription factor – 10
molecules per cell
8.1. Protein abundance in the cell
8.2. How much protein is needed and what level of purity is required?
Most sensitive instruments: Electrospray ionization (ESI)
Matrix-assisted laser desorption/ionization (MALDI)
0.2–1 pmol (25 kDa ~ 5–25 pg/ml)
Ion trap ~ 1fmol
8.2. How much protein is needed and what level of purity is required?
One mole of a protein is the amount that contains 6.023x1023 molecules of
that protein, which is known as Avogadro’s number. The weight of a mole of a
protein in grams (g) is the same as its molecular mass. For example, for a
protein with a molecular mass of 20,000 daltons, the weight of 1 mole of
protein is 20,000 g.
1 mmole = 10-3 moles 1 nmole = 10-9 moles 1 fmole = 10-15 moles
1 µmole = 10-6 moles 1 pmole = 10-12 moles
µ
µ
µ
µ
µ
µ
µ
8.3. Using recombinant proteins often simplifies the purification process
Purifying rare proteins from natural biological sources is often
extremely difficult, requiring extremely large quantities of starting
material and a 1–2-million-fold purification to achieve homogeneity
(growth factors, receptors or transcription factors).
µ
β β
β
8.3. Using recombinant proteins often simplifies the purification process
In contrast, purifying over-expressed recombinant proteins in
milligram to kilogram quantities has been greatly simplified by the
ability to produce target proteins containing a fusion partner (or “a
purification handle”) designed to facilitate protein purification.
TNF (E. coli) ? L medium Pkg. size: 50 µg – USD 625
TNF (HL60 tissue culture medium) 18 L – 20 µg of protein (Wang and Creasy, 1985)
The tumor necrosis factor (TNF-alpha) is a multifaceted polypeptide
cytokine known to be a mediator of inflammation and is an acute phase
protein which initiates a cascade of cytokines and increases vascular
permeability, thereby recruiting macrophage and neutrophils to a site of
infection. TNF- secreted by the macrophage causes blood clotting which
serves to contain the infection. TNF- has been detected in synovial fluid of
patients with rheumatoid arthritis.
8.4. Proteins can be separated on the basis of their intrinsic properties
Many proteins and peptides of biological interest are of very low
abundance, often constituting <0.1% of the total cellular proteins.
• Large quantities of source material required (>0.1%).
• Availability of separation facilities (e.g. instrumentation).
• Physical constraints of chromatographic resin support capabilities.
To fully exploit the chemical and physical properties of a target protein in
designing an appropriate strategy for its purification, the following parameters
for the target protein should be obtained in initial pilot studies:
• molecular weight (e.g., by SDS-PAGE, size-exclusion chromatography or
analytical centrifugation),
• pI (e.g., by isoelectric focusing), and
• stability with respect to pH, salt, temperature, proteases, inclusion of additives
to protein solvents to maintain biological activities (e.g. detergents, thiol
reagents, and metal ions).
8.4. Proteins can be separated on the basis of their intrinsic properties
8.4.1. Exposed amino acid side chains determine protein solubility
The protein-protein variation in solubility is due to the differences in the
ratio of solvent-exposed charged (i.e., polar) and hydrophobic amino
acids on protein surfaces.
Parameters that influence the solubility of a protein include:
solvent pH
the ionic strength and nature of the buffer ions
solvent polarity
temperature
Proteins tend to precipitate differentially from aqueous solution upon
addition of:
• neutral salts (ammonium sulfate)
• polymers (polyethylene glycol)
• organic solvents (ethanol, acetone)
Because it is not possible to predict with accuracy the solubility properties of
proteins, much of the skill in purification comes from experience in handling
proteins under a variety of conditions.
8.4. Proteins can be separated on the basis of their intrinsic properties
8.4.2. The size and shape of proteins affect their movement through liquids and gels
Titin is the largest known protein. Its human variant
consists of 34,350 amino acids, with the molecular weight
of the mature protein being approximately 3,816,188.13
Da. Its mouse homologue is even larger, comprising
35,213 amino acids with a MW of 3,906,487.6 Da.
Proteins vary markedly in size, ranging from a few amino acid residues of a few
hundred daltons to more than 10,000 amino acids with a molecular mass in excess
of 1,000,000 daltons.
However, the molecular mass of most proteins falls in the range of 6 kD to 200 kD.
In the purification techniques of
size-exclusion chromatography,
a protein solution is passed
through a column of porous beads.
The internal diameter of the pores
are such that large proteins do not
have access to the internal space
of the bead, whereas small
proteins have free access.
In SDS-PAGE, proteins are denatured and fully coated with the negatively
charged detergent SDS, such that they migrate in electrophoretic gels on the
basis of their molecular weight.
8.4. Proteins can be separated on the basis of their intrinsic properties
8.4.3. Differences in the surface charge of proteins are exploited in
ion-exchange chromatography
The net charge on a protein is the sum of the positively and negatively charged
amino acid residues, at the pH of the solvent.
Basic proteins (having net positive charge at neutral pH) have a majority
of basic amino acids (e.g., arginine, lysine, and histidine).
Acidic proteins (having net negative charge at neutral pH) have a
majority of acidic amino acids (e.g., aspartic acid, glutamic acid).
The pH at which the net charge of a protein is zero is referred to as the isolectric
point (pI).
8.4.4. Ligand-binding proteins may be purified by affinity
chromatography
Most proteins exert their biological function by specifically interacting with
some other cellular component. Enzymes bind to substrates, cofactors,
activators, inhibitors and metal ions.
Hormones bind to receptors.
Transcription factors bind to nuclear locations,
export signals and DNA templates.
8.4. Proteins can be separated on the basis of their intrinsic properties
The equisite specificity of antibodyantigen
interactions forms the basis
for immunoaffinity chromatography
Metal atoms attached to a
chromatographic support (immobilized
metal affinity chromatography IMAC)
8.4.5. Posttranslational modifications provide additional
opportunities for purification by affinity chromatography
8.4. Proteins can be separated on the basis of their intrinsic properties
Posttranslational modifications are fundamental to processes controlling
cellular behavior, including cell signaling, growth, and transformation.
Addition of carbohydrates to form glycoproteins
Addition of phosphates to form phosphoproteins
Addition of lipids to form lipoproteins
• Glycoproteins can be captured using immobilized
lectins.
• Phosphoproteins can be captured using
immobilized antibodies directed against
phosphotyrosine or, alternatively, using IMAC.
Leucoagglutinin, a toxic phytohemagglutinin
found in raw Vicia faba (Wikipedia)
8.4.6. Thermostable proteins can often be purified easily
8.4. Proteins can be separated on the basis of their intrinsic properties
Proteins are typically inactivated and precipitate if heated to 95°C.
However, some proteins exhibit a remarkable degree of thermoresistance:
Stathmin (mammalian intracellular regulatory protein)
Muscle phosphatase inhibitor I
Alkaline phosphatase (innate resistance to digestion with proteases)
Very often proteins with intrinsically
disordered structure are thermoresistant:
Tau (protein associated with
microtubules)
Casein (major milk major protein;
80%)
8.5. Devising strategies for protein purification
Before attempting to design a purification scheme, it is always worthwhile to
carry out pilot experiments on the crude extract to determine whether the
target protein possesses any unusual chemical properties that might be
exploited in a purification strategy.
Molecular weight
pI
Degree of hydrophobicity
Presence of carbohydrate (glycoprotein)
Phosphate modification
Free sulfhydryl groups
Stability with respect to:
• pH
• Salt
• Temperature
• Proteolytic degradation
• Mechanical shear
Bioaffinity for heavy metals
If the nucleotide sequence is known, much of this information might be
obtained by close inspection of the deduced amino acid sequence.
8.5. Devising strategies for protein purification
8.5.1. Is retention of biological activity essential?
An important consideration is whether it is essential to retain biological activity
of the target protein during purification.
Most proteins retain activity at:
• Low temperature
• Neutral aqueous buffers
• Stabilizing additives (glycerol, detergent)
Chromatography techniques use incompatible conditions:
• Organic solvents (acetonitrile)
• Ion-pairing acids (TFA)
• HCl (10 mM)
• NaOH (10 mM)
• MgCl2 (3 M)
• Glycine buffer (pH 2.3)
Reversed-phase column
Immunoaffinity columns
To limit the losses of biological activity of labile target proteins during purification, it is important to:
• minimize the number of steps in the purification protocol,
• avoid the need for buffer exchange between steps, and
• discriminate between losses of biological activity due to denaturation and physical losses
caused by irreversible adsorption to the chromatographic support or by proteolytic degradation.
8.5. Devising strategies for protein purification
8.5.2. How many purification steps are necessary?
The average number of steps necessary to purify proteins to homogeneity
is four, with an overall yield of 28% and a purification factor of 6380,
corresponding to an average ninefold purification and 73% yield per step.
It is generally recognized that with most conventional chromatographic supports,
there are compromises among speed, resolution, recovery, and capacity.
8.5. Devising strategies for protein purification
8.5.2. How many purification steps are necessary?
8.5. Devising strategies for protein purification
8.5.2. How many purification steps are necessary?
8.5. Devising strategies for protein purification
8.5.2. How many purification steps are necessary?
8.5. Devising strategies for protein purification
8.5.2. How many purification steps are necessary?
8.5. Devising strategies for protein purification
8.5.2. How many purification steps are necessary?
8.5. Devising strategies for protein purification
8.5.2. How many purification steps are necessary?
1. Clarifying the starting material
In purification protocol, it is always important to include steps for removing insoluble
residues (lipid droplets causing column blockage) and that the initial
clarification/concentration step be as rapid as possible (proteolytic degradation):
Differential centrifugation (awkward)
Filtration through a plug of glass wool or fine mesh cloth (less efficient)
Fractional precipitation (salts, polymers, organic solvents) – very high
(80%) average yield and able to gently concentrate large volumes
Ultrafiltration with a variety of molecular-mass cutoff limits (1,000–
300,000 daltons) – relatively slow
8.5. Devising strategies for protein purification
8.5.2. How many purification steps are necessary?
2. Capturing the target protein
For native proteins, enrichment is best accomplished using highcapacity/low-resolution
chromatographic procedures:
Hydrophobic interaction (HIC)
Anion-exchange chromatography
Nonbiospecific affinity chromatography (triazine dye
chromatography)
Immunoaffinity chromatography (high cost)
For recombinant proteins, several fusion systems are developed:
Oligohistidines (IMAC)
Antigenic epitopes (Mab)
Carbohydrate-binding proteins (domains recognized by lectins)
Biotin-binding domain (affinity to avidin or streptavidin)
If required, a specific protease cleavage site can be engineered into the fusion protein.
8.5. Devising strategies for protein purification
8.5.2. How many purification steps are necessary?
3. Purifying and concentrating-intermediate steps
This step should be designed to provide further purification and reduction
of sample volume, and it is best accomplished using intermediate
capacity/intermediate- to high-resolution chromatography.
1
2 3
4
5
8.5. Devising strategies for protein purification
8.5.2. How many purification steps are necessary?
4. Final polishing
The purpose of the final polishing step(s) is to remove any minor
contaminants remaining, to remove possible aggregates, and to prepare the
homogenous target protein for its intermediate use or for storage.
8.5. Devising strategies for protein purification
8.5.2. How many purification steps are necessary?
Which order of steps is best?
According to an analysis of succesful purification methods by Bonnerjea et al.
(1986):
homogenization
clarification/fractional precipitation
anion-exchange chromatography
affinity separation
SEC
An important consideration in designing the order of purification steps is to
minimize buffer-exchange steps:
Fractional precipitation using ammonium sulfate
Hydrophobic interaction chromatography
Ion-exchange chromatography
SEC, dialysis, or membrane ultrafiltration
8.5. Devising strategies for protein purification
8.5.2. How many purification steps are necessary?
Checklist for protein purification:
Define end goals
Establish a rapid analytical assay
In pilot experiments, define the chemical and physical
characteristic of target protein (pI, size, temperature stability,
ligand specificity)
Keep the purification procedure as simple as possible:
Minimize sample handling at every stage
Remove damaging contaminants
Be careful with addition of stabilizing
additives (detergent, salts)
8.5. Devising strategies for protein purification
8.5.2. How many purification steps are necessary?
8.5. Devising strategies for protein purification
8.5.3. Enrichment of low-abundance proteins by preparative electrophoresis
The dynamic range of protein abundance in a biological sample can be
1,000,000 copies per cell for the cytoskeletal proteins that maintain cellular
architecture. On the other hand, we can work with a transcription factor
ranging from 10 copies per cell.
2D electrophoresis can separate only a subset of a total proteome, at best
1,500–2,000 proteins.
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification
Chromatography (Greek: color writing) – technique for separating the
component of a mixture by allowing the sample to distribute between two
phases – one remains stationary (stationary phase), while the other moves
(mobile phase).
A packed bed of solid material in a column (liquid chromatography)
Spread as a thin layer or film on flat plat (thin-layer chromatography)
Paper (paper chromatography)
Liquid chromatography:
• A stationary phase (with controlled structure and surface chemistry).
• A column (packed with stationary phase).
• A mobile phase(s) or solvent(s) of controlled chemical composition
that moves the solute through the column.
• Chromatography equipment capable of accurately delivering the
sample to the column.
• Software programs for blending the mobile phase(s).
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification
8.5.4.1. Liquid chromatography – stationary phase
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification
8.5.4.2. Liquid chromatography – support matrix
Rigid solids – inorganic materials (porous silica, controlled pore glass,
hydroxyapatite, alumina and zirconium) (4,000–6,000 psi; 26–39 MPa)
Hard gels – synthetic organic polymers (polystyrene-divinylbenzene,
polyacrylamide, polyvinyl alcohols, and polymethacrylate)
POROS or SOURCE (2,000–5,000 psi; 13–35 MPa)
Soft gels – natural polymers such as cellulose, dextran, and agarose.
In most cases, support matrices used in protein and peptide applications
are hydrophilic, charge-neutral, and have low nonspecific binding
characteristics.
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification
8.5.4.3. Liquid chromatography – particle size and structure
Particle size (dp) – critical determinant in LC influencing the
chromatographic efficiency in a given separation (mechanical stability
– column lifetime; surface area – analyte capacity factor k´).
(POROS/SOURCE: dp = 3–20 m in diameter)
Particle shape – better to have narrow range of particle sizes
(packed columns with very broad particle distribution are inefficient
and less permeable)
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification
8.5.4.4. Liquid chromatography – pore structure (accessible surface
area)
Typically, the pore diameter must be 5 times the size of molecules being
purified to permit them to access all of the pores via molecular diffusion.
• Macroporus packings contain pores ranging from 1,000 to 10,000 Å (IEC).
• Mesoporous packings (wide-pore) contain pore diameters of 180–500 Å (HIC).
• Microporous packings have 60–120 Å pore diameters (RP-HPLC).
Stationary phase vs. bonded phase
Whereas the column packing matrix provides the chemically inert “skeleton”
for the stationary phase, the bonded phase provides the functional groups,
which are designed to selectively bind solute molecules.
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification
8.5.4.5. Liquid chromatography – bonded phase
Functional R groups are usually:
• Methyl (CH3) groups
• Hydrocarbon chains (C6, C8 or C18)
• Nitrile group (–C N)
• Amino group (–NH2)
• Carboxylic group (–COO-)
• Sulphate group (–SO3
-)
• Phenyl group (– )
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification
8.5.4.6. Liquid chromatography – chromatographic performance
The separation of charged molecules, such as peptides and proteins, as
they move down a column is affected by differential migration of solutes and
their spreading or dispersion (peak or band broadening).
When the interaction of a solute with the stationary phase is very strong, it
is retained to a greater extent, and thus will move more slowly.
Equilibrium distribution coefficient KD=SS/SM
SS – concentration of a solute in the stationary phase
SM – concentration of same solute in the mobile phase
The migration behavior is influenced by three major variables:
composition of the mobile phase (pH, ionic strength),
composition of the stationary phase, and
separation temperature.
Molecular spreading is the result of dilution of a solute band as it moves down
the column (kinetic and physical processes versus thermodynamic processes).
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification
8.5.4.7. Liquid chromatography – basic retention principles
Selectivity (α) is a measure of the
difference in retention between the solute
of interest and other solutes in the sample.
Retention is simply the time (tr) or volume
(vr) it takes for a solute to move through
the column.
The capacity factor (k´, or the retention
factor) is the number of column volumes
required to elute a particular solute, and t0
represents the void time.
k´ = (tr-t0)/t0
Selectivity is sometimes expressed as the ratio of the capacity factors k´ of two
solutes being separated: α = ´ ´
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification
N = theoretical plate number
Selectivity and retention
8.5.4.7. Liquid chromatography – basic retention principles
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification
8.5.4.7. Liquid chromatography – basic retention principles
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification.
Asymmetric peaks. A value >1
is a tailing peak (commonly
caused by sites on the packing
with a stronger than normal
retention of the solute).
Band broadening and efficiency
8.5.4.7. Liquid Chromatography – basic retention principles
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification
Resolution
Resolution (RS) is defined as the extent of separation between two
chromatographic peaks.
RS = 2 (trB – trA)/(WA + WB)
Resolution is a
composite function of
both thermodynamic
and kinetic parameters
and is expressed in
terms of an equation
that includes the
selectivity factor α, the
capacity factor k´, and
the plate number N.
RS = ¼(α−1)(N)½ [1/(1+k´)]
8.5.4.7. Liquid chromatography – basic retention principles
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification
Resolution
RS = ¼(α−1)(N)½ [1/(1+k´)]
k´ - Capacity is directly related to
the distribution coefficient of a
solute between the mobile and
stationary phases.
α - Selectivity is affected by the
surface chemistry of the column
packing, the nature and
composition of the mobile phase,
the nature of the stationary phase
and the gradient shape.
8.5.4.7. Liquid chromatography – basic retention principles
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification
Resolution
8.5.4.7. Liquid chromatography – basic retention principles
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification
8.5.4.8. Liquid chromatography – sample capacity
Sample capacity is the
amount of sample that can
be injected into a
chromatographic system
without overloading the
column (the number of
grams of sample per gram
of column packing).
• Measurement of saturation
or equilibrium capacity
• Frontal adsorption analysis
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification
8.5.4.8. Liquid chromatography – sample capacity
Column loadability
For optimal chromatographic
performance and to achieve
the greatest resolution, column
loadability is critical parameter.
8.5. Devising strategies for protein purification
8.5.4. Strategies based on chromatographic methods for protein
and peptide purification
8.5.4.8. Liquid chromatography – packing a column