Download Protein purification: the basics

Protein purification: the basics
Arvind Varsani
Reasons for protein purification
• To identify the FUNCTION of a protein
• To identify the STRUCTURE of a protein
• To use the use the purified product –
INTERMIDIATE- in downstream reactions
/ processing
• To produce a COMMERCIAL product
Selection of protein source
• Starting material can be from
– Animal tissue
– Plant material
– Biological fluids (e.g. blood, milk, sera)
RECOMBINANT expression
– Fermentation cultures (yeast, fungi, bacteria)
– Cell cultures (animal cells, plant cells, insect cells)
Important
• Protein in low concentration in natural
sources
– Need to induce expression
Or express recombinantly in various expression
systems
Classification of proteins by
structural characterisation
Structural characteristics
Examples
Comments
Monomeric
Lysozyme, growth hormone
Usually extracellular; often have
disulphide bonds
Oligomeric with identical subunits
Glyceralsehyde-3-phosphate
dehydrogenas, catalase, hexokinase
Mostly intracellular enzymes, rarely have
disulphide bonds
Oligomeric with mixed subunits
Petussis toxin
Allosteric enzymes, different subunits
have different functions
Membrane bound – peripheral
Mitrochondrial ATPase, alkaline
phosphatise
Readily solubilised in detergents
Membrane bound – intergral
Porins, insulin receptors
Requires lipid for stability
Membrane bound – conjugated
Glycoproteins, lipoproteins,
nucleoproteins
Many extracellular proteins contain
carbohydrate
Function
Examples
Amino acid storage
Seed proteins (e.g. gluten), milk proteins (e.g. caesin)
Structural – inert
Collagen, keratin
Structural – with activity
Actin, myosin, tubulin
Binding – soluble
Albumin, hemoglobin, hormones
Binding – insoluble
Surface receptors (e.g. insulin receptor), antigens (e.g. viral coat proteins)
Binding – with activity
Enzymes, membrane transporters (e.g. ion pumps
Relative abundance
Classification of proteins by
function
Protein properties and their effect on
development of purification strategies
Sample and target protein properties
Influence on purification strategy
Temperature stability
Need to work rapidly at lower temperatures
pH stability
Selection of buffers for extraction and purification (conditions for ion exchange, affinity or
reverse phase chromatography)
Organic solvents
Selection of condition for reverse phase chromatography
Detergent requirements
Consider effects on chromatographic steps and the need for detergent removal. consider
choice of detergent
Salt (ionic strength)
Selection of conditions for precipitation techniques and hydrophobic interaction
chromatography
Co-factors for stability or activity
Selection of additives, pH, salts and buffers
Protease sensitivity
Need for fast removal of proteases or addition of inhibitors
Sensitivity to metal ions
Need to add EDTA or EGTA in buffers
Redox sensitivity
Need to add reducing agents
Molecular weight
Selection of gel filtration media
Charge properties
Selection of ion exchange conditions
Biospecific affinity
Selection of ligand for affinity medium
Post translational modifications
Selection of group specific affinity medium
Hydrophobicity
Selection of medium for hydrophobic interaction chromatography
Yields for multi-step protein
purifications
100
•Limit the number of steps
•Optimise each step
•Be careful of the yield if
the proceduce requires
several steps
Key steps in purification
• Release of target protein from starting
material
• Removal of solids to leave the protein in
the supernatant
• Concentration of the protein
• Removal of contaminants to achieve the
desired purity
• Stabilization of the target protein
Three phase purification strategy
The final purification process should ideally consist of sample preparation,
including extraction and clarification when required, followed by 3 major
purifications step. The number of steps will depend on the purification strategy,
purity requirements and intended use of the protein
Protein analysis
• Tracking protein of interest and
determining the yield during purification
– Intended use of protein / source of starting
material
• Physical studies e.g. x-ray, NMR, EM
• End product – pharmaceuticals
Analysis of protein purity
• Total protein
• Specific quantification
– Activity assays
– Binding assays
• Detection of impurities
– HPLC
– Gel electrophoresis
• Protein mass spectrometry
Methods for quantification of
proteins in solution
Assay method
Useful range
NanoOrange assay
100ng/ml to
10ug/ml
BCA method (Cu
reduction)
0.5ug/ml to
1.5mg.ml
Bradford assay (dye
binding)
1ug/ml to 1.5
mg/ml
Lowry assay
1ug/ml to
1.5mg.ml
Absorbance at 280nm
50ng/ml to
2mg/ml
Comments
Samples can be read up to six hours later without any loss in the
sensitivity
Low protein to protein signal variability
Detection not influenced by reducing agents or nucleic acid
Samples must be read within 10 mins
Not compatible with reducing agents
Protein precipitates over time
High protein to protein signal variability
Not compatible with detergents
Lengthy, multi-step procedure
Not compatible with detergents, carbohydrates or reducing agents
High protein to protein signal variability
Detection influenced by nucleic acids and other UV absorbing
contaminants
BSA assay (Bicinchoninic acid)
• The first step is a Biuret reaction which reduces Cu+2 to Cu+1
• In the second step BCA forms a complex with Cu+1 which it
purple colored and is detectable at 562 nm
Bradford assay (coomassie dye
binding)
• Absorbance shift in Coomassie Brilliant Blue G-250 (CBBG)
when bound to arginine and aromatic residues
• The anionic (bound form) has absorbance maximum at 595 nm
whereas the cationic form (unbound form) has and absorbance
maximum at 470 nm
Lowry assay (Cu reduction)
The first step is a Biuret reaction which reduces Cu+2 to Cu+1
The second reaction uses Cu+1 to reduce the Folin-Ciocalteu
reagent (phosphomolybdate and phosphotungstate). This is
detectable in the range of 500 to 750 nm
Absorbance at 280nm
Monitors the absorbance of aromatic amino acids, tyrosine and
tryptophan or if the wavelength is lowered, the absorbance of the
peptide bond. Higher order structure in the proteins will influence
the absorption
Enzyme activity assays
• Continuous (kinetic assays)
– No separation step
• ELISA
• SDS
Cell disruption / breakage for
protein release
• Extraction techniques are selected based on the source of
protein (e.g. bacteria, plant, mammalian, intracellular or extra
cellular)
• Use procedures that are as gentle as possible. Cell disruption
leads to the release of proteolytic enzymes and general
acidification
• Selection of an extraction technique often depends on the
equipment availability and the scale of operation
• Extractions should be performed quickly, at sub-ambient
temperatures in a suitable buffer to maintain pH and ionic
strength
• Samples should be clear and free of particles before beginning
chromatography
Cell disruption: source variations
•
•
•
•
•
Tissues – variable
Mammalian cells – easy
Plant cells – some problems
Microbial cells – vary, common
Yeast and fungal cells – more difficult
Cell disruption: methods
•Chemical / enzymatic
•Cell lysis (osmotic
shock and freeze thaw)
•Enzymatic digestion
Blood cells
Mammalian
cells
•Fractional precipitation
•Extra cellular
proteins
•Mechanical
•Hand and blade
homogenizers
tissue
•Sonicator / disruptors
•Grinding with abrasive
plant/yeast
•Bad beaters / mill
•French press
•micro fluidizer
Lytic enzymes and detergents
• Lysozyme: disrupts bacterial cell walls
(hydrolysis of peptidoglycans) leading to cell
rupture
– Effective with gram positive bacteria, gram negative
generally require pre-treatment with a chelating agent
such as EDTA
• Detergents: anionic and non-ionic detergents
have been used to permeabilize gram negative
cells. Detergents are required for the release of
integral membrane proteins.
Simple shear methods
• Glass homogenizer (dounce, ten-broeck)
• sonicator
• French
pressure cell
Sample clarification
• Centrifugal sedimentation
• Coagulation and flocculation
• Filtration
Sedimentation
• Operates on the basis of density
difference between components in a
mixture (e.g. solids and liquid)
• Rate of sedimentation is dependent on:
– Magnitude of different in component densities
– Particle size, shape and concentration
– Magnitude of centrifugal force
– Flocculating of coagulating cells or organelles
Coagulation and flocculation
• Coagulation
– Increase in particle size from the joining of like
particles
– Promote by reducing charge repulsion
• Addition of multivalent ions (e.g. Al3+)
• Adjust pH to isoelectric point
• Flocculation
– increase in particle size by addition of agents acting
as bridges between particles
– Generally polyelectrolytes that neutralise surface
charges on particles and then link particles to form
aggregates
Concentration of extracts
•
•
•
•
Freeze drying
Dialysis
PEG precipitation
Concentration / fractionation by salting out
– Ammonium sulphate precipitation
• Ultraflitration
– Desalting
– Size fractionation
Protein purification
• Affinity chromatography
– Binding to immobilised ligands e.g antibodies, cofactors
• Ion exchange chromatography
– Anion (-) and cation (+) exchanger
• Hydrophobic interaction chromatography
– Colum coated with hydrophobic fatty acid chains
• Size exclusion chromatography
– Gel filtration
• Electrophoresis
– SDS