•
6.1. Raw material
Vertebrates:
• Liver studies – rat
• Skeletal muscle studies – rabbit
• Organs (heart, brain, kidney, thymus) – cow or pig
• Blood or placenta – human
Invertebrates:
• Are very small
• Difficult to dissect organ of interest from each individual
Plants:
• Spinach (Spinacia oleracea) chloroplast
• Beta vulgaris
Microorganisms:
• Yeast
• Bacteria
6.1.1. Species used for protein purification
6.1. Raw material
6.1.2. Freshness and storage
Yeast cakes remain viable: - weeks (0°C)
- month (frozen)
- years (sealed under
vacuum or in nitrogen)
Bacterial host cells: higher concentration of recombinant
protein production
- protein insolubility
- biologically inactive proteins
- protein processing
Figure 1. Growth of organisms in nutrient-rich medium. The ideal time for
harvesting is toward the end of the log phase before growth rate slows, giving
a high yield of cells. But specific enzymes may be maximum at an earlier or
later stage, so some trials at different times are desirable.
6.1. Raw material
6.1.2. Freshness and storage
6.1. Raw material
6.1.2. Freshness and storage
“Usually, the sooner the raw material is used, the better
and more physiologically relevant the preparation will be.”
•Natural degradative processes will have started.
Frozen storage:
• Free water freezes and ice crystals grow – very destructive
for membrane layers and organelles, not for protein.
• Decreasing temperature causes the remaining liquid to
become increasingly concentrated – solubility problem.
• pH changes drastically before complete solidification.
• Proteases liberated from lysosomes are activated.
• Freezing of extracts is sometimes preferable (proteolytic
enzyme inhibitors).
• Thawing speed can be important – the faster, the better.
6.2. Cell disintegration and extraction
Extraction of protein from tissues and cells is perhaps the most
critical step in any protein purification or proteomics strategy.
The principal aim:
• Achieve reproducibility.
• The highest degree of cell breakage.
• Minimal disruptive forces.
• Maintaining protein integrity (avoiding altering the
native structure – biological activity).
Perturbation of native protein structure can be caused by
exposure to:
• Extreme pHs,
• Extremes temperatures,
• Mechanical stress (shearing forces),
• Pressure, or
• Proteolytic degradation (association with host proteins).
6.2. Cell disintegration and extraction
Hand-operated
or motor driven
Waring blender Ultrasound
Vibrating bead mill Manton-Gaulin homogenizer
6.2. Cell disintegration and extraction
6.2. Cell disintegration and extraction
6.2.1. Mammalian tissues
• Frozen tissue stored below -50°C
• Dice tissues and cut away connective tissue and fat
• Extraction buffer: 2-3 vol/gram of tissue.
• Waring blender (30 s)
• Stir the homogenate (10-15 min)
• Check the pH
• Centrifuge (5,000-10,000 g/max 60 min)
• Decant extract through Miracloth
• Gentler conditions (Nonidet P-40), keep the sample cold, add protease
inhibitors
6.2. Cell disintegration and extraction
6.2.1. Mammalian tissues
6.2. Cell disintegration and extraction
6.2.1. Mammalian tissues
6.2. Cell disintegration and extraction
6.2.1. Mammalian tissues
6.2. Cell disintegration and extraction
6.2.1. Mammalian tissues
6.2. Cell disintegration and extraction
6.2.1. Mammalian tissues
6.2. Cell disintegration and extraction
6.2.1. Mammalian tissues
6.2. Cell disintegration and extraction
6.2.1. Mammalian tissues
6.2. Cell disintegration and extraction
6.2.2. Erythrocytes
• Collect red blood cells by centrifugation.
• Rinse with isotonic NaCl (0.9%, 0.15 M).
• Osmotic lysis with water (2 vol/1 vol packed cells).
• Selectively remove hemoglobin (90%) –
ethanol/chloroform.
Scanning electron micrograph
of blood cells. From left to
right: human erythrocyte,
thrombocyte (platelet),
leukocyte.
6.2. Cell disintegration and extraction
6.2.3. Soft plant tissues
• 0.5-1 vol of cold extraction buffer
• 20-30 mM of β-mercaptoethanol
• Homogenize in blender (30 s)
• Centrifuge as soon as possible (2-3 x 105 g min)
(minimize oxidative browning).
• Addition of powdered polyvinylpyrrolidone (for adsorbing phenols).
6.2. Cell disintegration and extraction
6.2.4. Yeasts
• Mechanical disruption (Manton-Gaulin homogenizer) 2-5 vol/gram
wet weight.
• Toluene autolysate (35-40°C) after 20-30 min.
• Ammonia cytolysis (0.5 M NH4OH) 2 vol/gram wet weight (16 -20 h).
Bring pH down (1-2 vol of water plus acetic acid).
• Bead mill (1 g/3 ml of buffer) “Merckenschlager”.
• Manual beading (glass beads of diameter 0.5, suitable bottle, shaken
manually for 5-10 min).
• Enzymatic lysis (mixture containing mannanases, glucanases and
chitinases).
6.2. Cell disintegration and extraction
6.2.5. Bacteria
• Sonication
• Dissolution of solids
• Dispersion of particulates in liquids
• Sonochemistry and chemical reaction acceleration
• Degassing of solutions
• Bead-milling (vortexing with glass beads)
• French press (8,000–20,000 psi [55-140 MPa], 10-30 ml)
• Grinding with abrasive agents (alumina or sand)
• Lysozyme (enzyme digestion 0.2 mg/ml + DNAase 10 ug/ml)
• Nitrogen cavitation – 800 psi (5.5 MPa) 1 – 1000 ml
Sonication https://www.youtube.com/watch?v=ykvS2gldQnw
6.2. Cell disintegration and extraction
6.2.5. Bacteria
Small-scale disintegration of
bacteria. Glass beads are
added to a small test tube (e.g.
15 mm diameter) to a depth of
about 10 mm. A slurry of cells
in buffer (e.g. 1 g wet weight in
5 ml of buffer) is added until up
to 5 mm above the glass
beads. The mixture is vortexmixed
for 2-5 min. The extract
is sampled from the surface of
the beads and centrifuged in a
microcentrifuge tube.
6.2. Cell disintegration and extraction
6.2.5. Bacteria
A different problem can occur with E. coli expression system,
where the expressed recombinant protein appears in the crude
extract:
• Insoluble aggregates – “inclusion bodies” need to be
solubilized in a strong denaturant (e.g. Guanidin
hydrochloride or urea).
• Lysis conditions will depend on whether the lysate is to be
used for:
• Immunoprecipitation studies,
• Western blotting,
• 2D electrophoresis,
• Native target protein isolation using conventional chromatographic
purification procedures, or
• Recombinant protein purification procedures that rely on the target
protein expressed as fusion protein including a purification “handle”
or “tag”.
6.2. Cell disintegration and extraction
6.2.5. Bacteria
6.2. Cell disintegration and extraction
6.2.5. Bacteria
6.2. Cell disintegration and extraction
6.2.5. Bacteria
6.2. Cell disintegration and extraction
6.2.5. Bacteria
6.2. Cell disintegration and extraction
6.2.5. Bacteria
Inclusion body formation can be prevented and the soluble fraction of
the target protein increased by:
• Lowering temperature from 37°C to 30°C, 22°C or even lower,
• Varying the media composition and using different strains,
• Co-expression of molecular chaperones,
• Fusion of the target protein with a highly soluble protein, or
• Growing cells in the presence of sorbitol and glycyl betaine.
Two major advantages of inclusion bodies:
• By sequestering recombinant protein, these bodies permit the cell to
express the protein at high levels.
• The inclusion bodies can be readily purified away from bacterial
cytoplasmic proteins by centrifugation.
6.2. Cell disintegration and extraction
6.2.5. Bacteria
6.2. Cell disintegration and extraction
6.2.6. Fatty tissues
• Homogenization difficulties
• Large amounts of detergent
• Acetone powder
6.3. Optimization and clarification of the extract
• Adjust the acidification (cause the aggregation).
• The best method is to make small-scale extract using a large volume
(10ml/g) of extractant, and measure the amount of activity after different
times of treatment.
• Isoosmotic buffer – for isolating organelles (sucrose, mannitol or sorbitol).
• EDTA (1–5 mM) destabilize complexes and inhibit proteases.
• β-mercaptoethanol (reducing condition).
• Typical buffers: 20-50 mM phosphate, pH 7–7.5; 0.1 M Tris-HCl, pH 7.5;
0.1 M KCl with a little buffer in it.
• Ionic strength inside (0.15–0.2 M).
6.3. Optimization and clarification of the extract
Provided that the protein wanted, acidification can be most
beneficial if:
1. does not also isoelectrically precipitate at the pH used,
2. does not adsorb to the precipitate forming, and
3. remains stable at the subphysiological pH.
The extract should be:
• kept cold,
• controlled for pH after stirring 10-20 min,
• readjusted after pH measurement.
• Plant tissues - are more acidic.
• Microorganisms - contain large amount of nucleic acid.
- The cell wall and extracellular material may either
be finely dispersed (turbid extract) or partially
solubilized (gum-like polysaccharide in solution).
A common procedure is to treat with substances that cause
precipitation of the nucleic acids and associated compounds. These
include:
• Streptomycin (precipitates ribonuclear proteins and clarifies the
extract)
• Protamine (precipitates DNA and RNA complexes)
• Polyethyleneimine (precipitates nucleic acids and causes
precipitation of aggregated nucleoproteins)
6.3. Optimization and clarification of the extract.
6.4. Extraction of membrane proteins
Many proteins and enzymes are not naturally present in an
aqueous phase:
• cell membrane proteins in prokaryotes;
• in gram-negative organisms, periplasmic proteins
immobilized between the outer and inner membranes;
• in multicellular eukaryotes, individual cell membranes
and organelles (mitochondria, nuclei, endoplasmic
reticulum, Golgi, vacuolar and lysosomal membranes).
Membrane proteins are categorized as:
• peripheral (loosely associated on the surface of the
membrane) or
• integral.
6.4. Extraction of membrane proteins
How to release membrane proteins as soluble:
1. Sonication
2. EDTA, EGTA at 1-10 mM
3. Mild alkaline conditions (pH 8-11) at low ionic strength
4. Dilute non-ionic detergent
5. Low concentrations of partially miscible organic solvents such as n-
butanol
6. High ionic strength, e.g., 1 M NaCl
7. Phospholipase treatment
Some proteins have hydrophobic patches on the surface which will mutually
attract, resulting in aggregation.
Some detergents commonly used
for extraction of membrane proteins.
In general, the ionic detergents are
more solubilizing, but also are more
likely to denaturate the solubilized
proteins.
Nonionic:
• Tween 20
• Tween 80
• Triton X-100
• Triton X-114
• Emulgens
• Lubrol
• Digitonin
• Octyl glukoside
Zwitterionic:
• Lysoletcithin
• CHAPS
• CHAPSO
• Zwitergents
Ionic:
• Cholate
• Deoxycholate
• CTAB
• SDS – dodecyl sulfate
6.4. Extraction of membrane proteins
6.4. Extraction of membrane proteins
• 5 mg/ml protein suspension requires at least 1% of detergent.
• All detergents form micelles.
• Micelle clusters range from 30,000 to 100,000.
• Triton X-114 extracts membrane proteins at close to 0°C using 1-3% detergent.
• Chloral hydrate in 100% aqueous w/v.
Chloral hydrate is produced from chlorine and ethanol in acidic solution.
In basic conditions, the haloform reaction takes place and chloroform is
produced.
4 Cl2 + C2H5OH + H2O Cl3CCH(OH)2 + 5 HCl
6.5. Differential detergent fractionation
Differential detergent extraction is an established method for cell
fractionation, which partitions subcellular constituents into functionally
and structurally distinct compartments.
Differential detergent fractionation (DDF) applicable for fractionation:
• cell grown in suspension
• monolayer culture
• whole tissues
• further subfractionation
DDF preserves the structural and functional integrity of cellular proteins and
is useful in a variety of proteome research applications:
• Determine the subcellular localization of biological proteins.
• Semi-purify compartment-specific macromolecules.
• Enrich for low-abundance proteins.
• Investigate dynamic interactions between cytosolic and structural
entities (membranes bind the cytoskeleton).
• Monitor treatment-induced compartmental redistributions of
macromolecules.
6.5. Differential detergent fractionation
6.5. Differential detergent fractionation
6.6. Preparation of subcellular extracts
The aim of subcellular fractionation is to separate
cellular compartments with minimal damage.
• Homogenization of tissues and cells followed by separation of
cellular organelles:
• achieves maximum cell breakage in a reproducible
manner,
• uses disruptive forces that minimize damage to the
organelles of interest, and
• retains the original structure and functional integrity of the
organelles of interest.
The principal methods for disrupting cells (osmotic shock,
ultrasonic vibration, mechanical grinding or shearing, and nitrogen
cavitation):
• Centrifugal methods that separate organelles by size
• Immunoisolation methods that use antibodies
• Electrophoresis methods that separate proteins on the basis of
surface charge distribution
6.7.1. Lysis of cultured cells for immunoprecipitation
6.7. Sample protocols
6.7.1. Lysis of cultured cells for immunoprecipitation
6.7. Sample protocols
6.7.1. Lysis of cultured cells for immunoprecipitation
6.7. Sample protocols
6.7.2. Lysis of cultured cells for immunoblotting
6.7. Sample protocols
6.7.2. Lysis of cultured cells for immunoblotting6.7. Sample protocols
6.7.3. Disruption of cultured cells by nitrogen cavitation
6.7. Sample protocols
6.7.3. Disruption of cultured cells by nitrogen cavitation
6.7. Sample protocols
6.7.3. Disruption of cultured cells by nitrogen cavitation
6.7. Sample protocols
6.7.3. Disruption of cultured cells by EmulsiFlex-C3
6.7. Sample protocols
The EmulsiFlex-C3 homogenizer
is powered by an electric motor.
There are no "O"-rings or gaskets
in the entire path of the product.
No compressor is needed to run
the pump.
6.7.3. Disruption of cultured cells by Pressure Cell Homogenizer
6.7. Sample protocols
https://www.youtube.com/watch?v=b9-G-n4crpk
• Continuously adjustable proces pressure from
1,500 psi (10 MPa) to 60,000 psi (410 MPa)
allows processing of ingredients that are difficult
or uneconomical to process in any other way,
offering the widest scope of formulations
• The homogenising valve features ceramic needle
and seat with profiled geometry for optimised
performance. Pneumatically controlled, it provides
constant pressure and allows fine adjustment
• Choice of (easily interchangeable) processing
cells: 1 ml, 10 ml, 18 ml and 35 ml
• The inverted design and full-stroking piston with
bore seal practically eliminates dead volume
• Can be run in single-shot mode - including very
viscous formulations - and continuous mode
(except the 1 ml cell). The flow rate in both modes
is adjustable from drop-by drop to 200 ml/min
(depending on the processing cell and formulation)
• All wetted parts are simply removable without
tools in less than one minute for cleaning,
sterilization (including autoclave), pre-cooling, or
pre-warming, all as required by the
sample/process
• Three options for temperature control