Protein purification: the basics

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Protein purification the basics

• Arvind Varsani

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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

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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)

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Important

• Protein in low concentration in natural sources
• Need to induce expression
• Or express recombinantly in various expression
  systems

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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
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Classification of proteins by function
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
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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
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Yields for multi-step protein purifications

• Limit the number of steps
• Optimise each step
• Be careful of the yield if the proceduce requires
  several steps

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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

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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
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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

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Analysis of protein purity

• Total protein
• Specific quantification
• Activity assays
• Binding assays
• Detection of impurities
• HPLC
• Gel electrophoresis
• Protein mass spectrometry

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Methods for quantification of proteins in solution
Assay method Useful range Comments
NanoOrange assay 100ng/ml to 10ug/ml 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
BCA method (Cu reduction) 0.5ug/ml to 1.5mg.ml Samples must be read within 10 mins Not compatible with reducing agents
Bradford assay (dye binding) 1ug/ml to 1.5 mg/ml Protein precipitates over time High protein to protein signal variability Not compatible with detergents
Lowry assay 1ug/ml to 1.5mg.ml Lengthy, multi-step procedure Not compatible with detergents, carbohydrates or reducing agents
Absorbance at 280nm 50ng/ml to 2mg/ml High protein to protein signal variability Detection influenced by nucleic acids and other UV absorbing contaminants
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BSA assay (Bicinchoninic acid)

• The first step is a Biuret reaction which reduces
  Cu2 to Cu1
• In the second step BCA forms a complex with Cu1
  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

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Lowry assay (Cu reduction)
The first step is a Biuret reaction which reduces
Cu2 to Cu1 The second reaction uses Cu1 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

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Enzyme activity assays

• Continuous (kinetic assays)
• No separation step

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• ELISA
• SDS

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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

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Cell disruption source variations

• Tissues variable
• Mammalian cells easy
• Plant cells some problems
• Microbial cells vary, common
• Yeast and fungal cells more difficult

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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

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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.

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Simple shear methods

• Glass homogenizer (dounce, ten-broeck)

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• French pressure cell

• sonicator

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Sample clarification

• Centrifugal sedimentation
• Coagulation and flocculation
• Filtration

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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

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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

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Concentration of extracts

• Freeze drying
• Dialysis
• PEG precipitation
• Concentration / fractionation by salting out
• Ammonium sulphate precipitation
• Ultraflitration
• Desalting
• Size fractionation

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Protein purification

• Affinity chromatography
• Binding to immobilised ligands e.g antibodies,
  co-factors
• Ion exchange chromatography
• Anion (-) and cation () exchanger
• Hydrophobic interaction chromatography
• Colum coated with hydrophobic fatty acid chains
• Size exclusion chromatography
• Gel filtration
• Electrophoresis
• SDS