Transcript Chapter 5 Cell disintegration and extraction techniques

Chapter 5
Cell disintegration and
extraction techniques
Overview
To get the intracellular product
the disintegration of cells is need
The methods used to break cells depend largely on the fragility of the cells
ex) animal cells: burst by osmotic shock, freeze/thaw, enzyme digestion(lipase, protease), toluen
plant tissue: pectinase and cellulase treatment
microbial cells: lysozyme treatment
To achieve a good yield,
1. minimize the number of steps
2. choose appropriate disruption methods
(a) can a given disruptor be used for a particular cell type?
(b) which is the best method of extracting a product?
Key questions
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Stability of the released protein
Location of target protein within the cell
The yield and kinetics of the process
Continuous or batch disruption
The need to consider subsequent steps
Assesing the extent of disruption
Marker substance for cell disruption
Containment of the process
-avoid release of harzardous intracellular products into envronment
9. Scale-up-cost, volume, sample viscosity
1.Stability of the released protein
•The disruption methods can impose great physical and chemical stress
ex) heat generated by mechnical disruption may result in Nz denaturation
activation of proteolytic enzyme can degrade target Nz
•Consideration: minimize the stress condition
3.The yield and kinetics of the process
•Yield-the quantity of enzyme released/unit starting materials
•Specific activity-the amount of enzyme(unit)/ released protein(g)
•Factors affecting the yield of enzyme
(a) location of product with in the cell
(b) degree of disintegration
(c) extent of denaturation of the product during disruption
•Disruption rate can be modelled by “First-order process”
R=RM(1-e-kt) : time-dependent
consider the “optimun disruption time”
5. The need to consider subsequent steps
•After disruption, clarification step is need for subsequent purification step
usually, through centrifugation
in the laboratory, high g force
on larger scale, clarifying cell lysate is difficult
 whether a laboratory centrifuge is capable of sedimenting cell debris
t=
In(RS/RL) *18μ
ω2(ρP-ρL)dP2
t: the centifugal time
RS:radii from centrifuge tube head to liquid surface
RL:radii from centrifuge tube head to bottom
μ:liquid velocity
ρP:particle density
ρL:liquid dencity
ω:angular velocity =2π(rpm)/60
dP:mean diameter of cell debris
•Mechanical disruption methods are not needed chemicals that intetfere with
subsequent purification step
6. Assesing the extent of disruption
•Use a marker substance (estimate total intracellular proteins)
•specific activity could decrease during disruption
because of releasing of non-product proteins and denaturation of enzyme during disruption
7. Marker substance for cell disruption
to determine the degree of cell disruption, marker techniques are used
1. Biological: visible cell counting
2. Physical: measuring the Vol. of intact cells, O.D., viscosity of the sample
3. Chemical: measuring the protein concentration
Methods of disruption
4℃
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•Slow the rate of enzyme damage caused by proteas(add PMSF in eukariotic cell)
•Prevent thermal denaturation
Pre-treatment of material
General procedure notes
Mixers and blenders
Coarse grinding: pestle and mortar
Fine grinding:the bead mill 10. Osmotic shock
Homogenization
11. Lytic enzymes
Ultrasonication
12. Chemical treatment
Heat shock
13. Detergents
Freezing and thawing
14. Slovents
1. Pre-treatment of material
To increase the degree of cellular disruption
Combine two(or more) methods
for example, freeze-thaw step or pre-treatment of solvents or detergents
before mechnical disruption
2. General procedure notes
Table 1.The main cell disruption methods
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Methods needing specialist equipment
(a) mixer and blender
(b) coarse grinding with pestle and mortar
(c) fine grinding in a bead mill
(d) homogenization
(e) ultrasonication
Disadvantage:Cells are not extensively distintegrated
incubation time
operational parameters
(conc.of lysing chemicals, bead size, conc.,
agitator speed for bead mill, pressure for homogenization,
Vol. Of sample for ultrasonication)
Methods using non-specialist equipment
(a) freezing and thawing
(b) osmotic shock
(c) chaotrophic agents
Disadvantage: addition of extra chemicals to
(d) detergents
the system may interfere with downstream processing
(e) solvents
(f) enzyme lysis
3. Mixers and blenders
•Grind cells coarsely
•Use the buffer containing inhibitors, reducing agents(see protocol 2)
4. Coarse grinding: pestle and mortar
•Useful for disruption of tissue samples
•Samples were grinded to fine powder under liquid nitrogen
•Nacessary to maintain the frozen state
5. Fine grinding: the bead mill
•Useful for disruption of micro-orgarnisms
•Bead mills should have cooling jackets
because of heat generation during disruption
•Optional parameters: bead size, bead volume, agitator speed, milling time
6. Homogenization
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Hand-held piston/plunger device
animal cells are easily disrupted, but very inefficient
High-pressure homogenizer
suitable for large scale operation
principles;
sample
narrow orfice High pressure cell crusing
pumping
cell breaking
usually, increasing the pressure increase the amount and rate of protein release
according to k=k’Pn(first-order reaction, p98)
High-pressure homogenizer
Mechanical homogenizer
7. Ultrasonication
•Very vigorous process, results in complete solubilization
•Principles;
the vibrating titanium probe creat cavities
collaps of the cavities
pressure changes and shear foreces which cause cell disruption
•Problem: heat-generation(cause thermal denaturation, alteration of Nz activity
impossible to adjust the power input
•The release curve for protein is usually first-order(Fig. 4), but could be affected by
power input, sample volume
•Very useful for fragmentaion of cellular DNA related with increaseing of viscosity
Non-mechanical disruption methods
Heat shock
•Consider the thermal denaturation. Leading to loss of Nz activity
•Useful for purification of heat-stable proteins(e.g.ubiquitin)
Freezing and thawing
•Very simple, but suitable for cells without a cell wall
•Repeating of freezing and thawing may cause denaturation of proteins
Osmotic shock
Lytic enzymes – bacterial disruption with lysozme
useful for Gram positive cells
Chemical treatment
·dvantage: the cell will be left substantially intact
· chemicals must bo compatible with further downstrea process
· chelating agents(sequest divalent cation)and chaotropic agents(weaken hydrophobin interaction)
Detergents-increase protein solubility
Slovents – dosen’t inactivate the enzyme products
toluene, ether, isoamylalcohol, chloroform
Colclusions: choice of methods
(a) The mechanical methods of cell disruption have the widest
application for laboratory and pilot scale disruption
(b) Homogenization has proved an effective large scale process
(c) Chemical methods are, generally, cell/product specific and
thus, not applicable to all system