Proteins Module - Biochemistry 1st year

Protein Purification and Characterisation

Why purify a protein?
Protein Purification Isolate the protein
1. Protein sources & extraction  identify function, specificity,
2. Purification by precipitation kinetics etc.
3. Purification by Chromatography  identify the structure
4. Designing & assessing purification schemes  as a reagent
 diagnostic or therapeutic e.g.
Factor VIII from plasma
What is a protein?
 Proteins are vital to life
 Polypeptide, functional groups
 Folds into 3D structure - Hydrophobic collapse, non-covalent interactions
 Unique properties - Charge, hydrophobicity, affinity etc.
 Exploit in purification




How pure is pure? – defined by
the end use (only remove
contaminates that interfere)




Source of Protein

Natural sources

 Required protein MUST be abundant, stable, readily available
 Wide range - blood (Factor VIII), milk, plant or animal tissues. e.g. 10-15mg hydrolytic enzymes from
200g kidney

Advantage: the proteins are in the in vivo form
Disadvantages: the desired protein is often in low abundance and reproducibility

Plant sources: low abundance (polysaccharides)
 oxidation
 seeds: high levels storage proteins
 fruit: developmental changes

Recombinant sources
 clone gene, transfer to plasmid, overexpress
 recombinant cells. e.g. bacteria, yeast, insect, plant cells, transgenic animals
 yields may be 100-fold higher
 Most successful in similar organism - with deleted or low native expression

Disadvantage – depends on the source
Prokaryotic expression

,• under control of strong promoter
• expression induced
• insolubility - necessitates solubilisation and refolding - not always successful, but purification method

e.g. E.Coli




Eukaryotic expression
 Yeast (S. cerevisiae)
 Pichia pastoris - higher cell density, grows on methanol, still misfolds
 Insect cells - baculovirus driven expression

A d v a n ta g e s D is a d v a n ta g e s
G o o d .g ro w th .ra te s m is f o ld i n g
s im p l e . ( c h e a p ) . m e d ia h y p e r g ly c o s y l a t i o n
g e n e t ic s . u n d e r s t o o d n o t . h ig h . c e l l. d e n s i t y

 mammalian recombinant expression - mammalian transgenic expression (milk)




Extraction of protein
 Homogenisation
 Cell lysis - Physical methods, Non-mechanical methods
 Subcellular fractionation

Homogenisation (natural sources)

Mammalian tissue
 cut into small pieces, then blender - isotonic buffer e.g. 0.3M sucrose
 ease dependent on tissue connectivity - e.g. lung more difficult than brain, kidney

Plant tissue
 blender for fragile tissues, e.g. leaf,
 grinding with sand for fibrous sources e.g. stems,
 e.g. dry grind first (freeze in N2(liq)) if protein stable

Cell Lysis - Physical or non-mechanical methods
 Breaking of plasma membrane (cell death):
 Easiest for eukaryotic cells
 Releases cell contents
 Proteins AND proteases, nucleic acids
Physical cell disruption

Mechanical Sonication:
 high frequency sound waves
 oscillating metal probe

, rotating blades e.g. Waring blender
 grinding in pestle & mortar
 vortexing with glass beads

Liquid shear
 e.g. French Press homogenizer
 suspension forced through narrow space
 Pressure release bursts cells
 only small volumes, difficult to clean


Non-mechanical lysis

osmotic lysis - hypotonic solution, insect, mammalian cells

freeze/thaw - ice crystals disrupt cells

detergent solubilisation e.g. BugBuster® - non-ionic (ionic e.g. SDS denature)

Lytic enzymes damage cell walls - Followed by osmotic shock, application specific e.g. lipases, proteases




 May be combined
 Need to preserve activity
 Medium: buffer, salts, EDTA, protease inhibitors, detergents, etc.
 Needs optimisation
 Chill to 4°C
Subcellular Fractionation
 eukaryotic cells
 gentle disruption retains compartments
 differential centrifugation (size)
o components retain activity
o cytosol (soluble small proteins)




Protein produced from native or recombinant sources
as in last lecture. Cells lysed, proteins removed from cell debris by centrifugation, now need to
separate protein of interest from host cell proteins, nucleic acids and other biomolecules. Protein
purification is the removal of contaminants.

, Clarification
 Remove cell debris, other particulates
 Filtration - Can lower protein yield
 Sedimentation - Centrifugation (CRP lectures), large or small scale
 Supernatant: soluble proteins
 Pellet: Membrane fraction, organelles….

Contaminants – particulates, aggregates, nucleic acids, small molecules, proteins

Removal of nucleic acids
 Filtration - Gelatinous/fibrous so clog
 Precipitate with 1% protamine sulphate (+ve) - Simple, rapid, add contaminant, poor reproducibility
 Incubate with DNase, (30 min, 25-37°C) - Simple, reproducible, Slow, proteases may degrade target
 Extract with sonication (shears chromosomes)

Removal of small molecules
 (buffer exchange)
 Ultrafiltration - Semi-permeable membranes - define MW cutoff
 Dialysis


Proteins
 general host proteins
 variants of target protein

Polymer of amino acids
Exploit chemical and physical properties
 Charge (isoelectric point, surface charge)
 Hydrophobicity
 Affinity (activity, metal ions) interactions and binding
 Solubility/Stability
 Molecular weight



Purification by exploiting solubility - Protein or contaminants

Purification by precipitation

Hydrophobic regions interact with water – no H bonds formed. An
order around the molecule.

Solubility: polar (solvent), ionic (salts), repulsive
Reduce solubility, selective precipitation

Precipitation by changes in ionic strength – differential
solubilisation

1. ionic strength
Increase from 0M - Differential solubilisation, ‘salting in’ to solution.