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Downstream Processing - Protein Extraction and Purification

Downstream Processing - Protein Extraction and Purification

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Chapter 17



462



bacterium Escherichia coli, and is completely free from the unwanted

prion.

Enzymes or proteins produced by microorganisms may be intracellular, periplasmic or secreted into the culture medium. For extracellular

enzymes, the degree of purification required is often minimal, as the final

product is intended for industrial use and does not have to be of high

purity. Such large-scale processes may yield tonnes of protein product.

Many other enzymes are produced in more complex intracellular

mixtures and represent a greater challenge to the protein purification

scientist. Those produced for therapeutic use must attain very high and

exacting standards of purity and to achieve this it may be necessary to

develop complex purification protocols. Again, recombinant DNA

technology has been able to help in this area. In the case of therapeutic

proteins, there are often advantages if the protein of interest can be

secreted, either into the periplasm or into the medium. This results in an

enormous reduction in the level of contaminating proteins and other

macromolecules, such that pure product can often be obtained in only

two or three steps of purification. Choice of a suitable expression system

can result in higher yields in the fermenter, thereby improving the specific

activity of the starting material. It is also possible to add groups to a

protein which aid its purification by conferring specific properties and

subsequently removing these groups when they are no longer required.

In designing a large-scale purification process the number of steps, and

the recovery of product at each step can have a major effect on the overall

yield of the product, as is shown in Figure 1. The typical recovery of a

chromatographic step is between 80% and 9070,so a complex purification requiring many steps may have an overall yield as low as 10% of the

starting material. This may not matter for a laboratory-scale purification, but for large-scale production it is important to optimize the overall

process from expression system or fermentation to final polishing step,

so as to minimize the number of purification steps required.



2



CELL DISRUPTION



There are three main methods for the release of intracellular proteins

from microorganisms: enzymic, chemical or physical. Not all of the

techniques available are suitable for use on a large scale. Perhaps the

main example is sonication, which is frequently the method of choice for

the small-scale release of proteins. On a large scale it is difficult to

transmit the necessary power to a large volume of suspension and to

remove the heat generated.



Downstream Processing:Protein Extract ion and Pur iJicat ion



463



100



80

Recovery per step



m

5



60



9546



F

-



E



8



40

90%



20



85%



80%

I



0



0



2



I



I



I



4



6



8



10



Number of Steps



Figure 1 The eJtrect of recovery per step on overall recovery



2.1



Enzymic Methods of Cell Disruption



Lysozyme, an enzyme produced commercially from hen egg white,

hydrolyses beta- 1,4-glycosidic bonds in the mucopeptide of bacterial

cell walls. Gram-positive bacteria, which depend on cell wall mucopeptides for rigidity are most susceptible, but final rupture of the cell wall

often depends upon the osmotic effects of the suspending buffer once the

wall has been digested. In Gram-negative bacteria lysis is rarely achieved

by the use of lysozyme alone, but the addition of EDTA to chelate metal

ions will normally result in lysis. Although gentle, this technique is rarely

used for the large-scale extraction of bacterial enzymes, perhaps due to

the relatively high cost of lysozyme and the possibility of introducing

contaminants. It has been used for the large-scale release of an aryl

acylamidase from Pseudomonas fluorescens.



'



2.2 Chemical Methods of Cell Lysis

2.2.1 Alkali. Treatment with alkali has been used with considerable

success in small- and large-scale extraction of bacterial proteins. For

example, the therapeutic enzyme, L-asparaginase, can be released from

Erwinia chrysanthemi by exposing the cells to pH values between 11.0



' P. M.Hammond, C. P. Price, and M. D. Scawen, Eur. J . Biochem., 1983, 132,651.



464



Chapter 17



and 12.5 for 20 min.2 The success of this method relies on the alkali

stability of the desired product. The high pH may inactivate proteases,

and the method is of value for the combined inactivation and lysis of

rDNA microorganisms.



2.2.2 Detergents. Detergents, either ionic, for example, sodium lauryl

sulphate, sodium cholate (anionic) and cetyl trimethyl ammonium

bromide (cationic), or non-ionic, for example Triton X-100 or X-450, or

Tween, have been used to aid cell lysis, often in combination with

lysozyme. Ionic detergents are more reactive than non-ionic detergents,

and can lead to the denaturation of many proteins. The presence of

detergents can also affect subsequent purification steps, in particular salt

precipitation. This can be overcome by the use of ion exchange

chromatography or ultrafiltration, but obviously introduces additional

steps.



2.3 Physical Methods of Cell Lysis

2.3.1 Osmotic Shock. Osmotic shock can be used for the release of

enzymes and proteins from the periplasmic space of a number of Gramnegative bacteria. The method involves washing the cells in buffer

solution to free them from growth medium, and then suspending them

in 20% buffered sucrose. After being allowed to equilibrate, the cells are

harvested and rapidly resuspended in water at about 4°C. Only about 48% of the total bacterial protein is released by osmotic shock, and if the

required enzyme is located in the periplasmic region it can produce a 14to 20-fold increase in purification compared with other extraction

techniques. A major disadvantage of osmotic shock is the large increase

in volume which occurs.

2.3.2 Grinding with Abrasives. Initially this technique was restricted

to the grinding of cell pastes in a mortar with an abrasive powder, such as

glass, alumina or kieselguhr. It has since been developed and mechanized

using machines originally developed for the wet grinding and dispersion

of pigments in the printing and paint industries. A typical product, the

Dynomill (W. A. Bachofen, Switzerland) can be used to release proteins

from a wide variety of microorganisms. It consists of a chamber containing glass beads and a number of fixed and rotating impeller discs. The cell

suspension is pumped through the chamber, and the rapid agitation is

sufficient to break even the toughest of bacteria. The disintegration

2T. Atkinson, B. J. Capel, and R. F. Sherwood, in ‘Safety in Industrial Microbiology and

Biotechnology.’, ed. C. H. Collins and A. J. Beale, Butterworth, Oxford, 1992, p.161.



Downstream Processing: Protein Extraction and PuriJication



465



chamber must be cooled to remove the heat which is generated. A

laboratory-scale model, with a 600 ml chamber can process up to 5 kg

bacteria per hour, and production scale models are available with

chambers of up to 250 litre capacity.

Many factors influence the rates of cell breakage, such as the size and

concentration of the glass beads, the type, concentration and age of the

cells, chemical pre-treatment, the agitator speed, the flow rate through

the chamber, the temperature, and the arrangement of the agitator discs,

and these have been investigated for yeasts3 and b a ~ t e r i a .This

~ - ~ type of

cell disrupter has the advantage that it can be readily mounted in an

enclosed cabinet when pathogenic or rDNA organisms are to be broken.

2.3.3 Solid Shear. Methods of cell disruption employing solid shear

have long been used on a small scale. It involves the extrusion of frozen

cell material through a narrow orifice at high pressure and an outlet

temperatures of about -20°C. It has found little application on an

industrial scale, due to limitations on the amount of material which can

be processed.



2.3.4 Liquid Shear. Liquid shear is the principle choice for the largescale disruption of microbial cells, finding widespread application in

both industrial processes and in research. It is particularly useful for the

disruption of bacteria and yeast?

As with solid shear, the cells are passed through a restricted orifice

under high pressure, this time in a liquid suspension. For smaller scale

work, a French Press is used. Larger scale work usually employs a

homogenizer of the type developed for emulsification in the dairy

industry. A temperature increase of at least 10°C in a single pass is not

uncommon and it is necessary to pre-cool the cell suspension before

homogenization. The liquid shear homogenizer is normally operated at

wet cell concentrations of about 20%.

For large-scale work the Manton-Gaulin homogenizer (APV Ltd.,

Crawley, UK) is the most frequently used. It consists of a positive

displacement piston pump with a restricted outlet valve, which can be

adjusted to give the required operating pressure, up to 95 MPa. The

smallest Manton-Gaulin homogenizer, the 15M-8TA, has a throughput

of about 50 1 h-' at a pressure of 55 MPa. A larger version, the MC-4,

has a throughput of about 300 1 h-', again at a pressure of 55 MPa.

The rate of cell breakage and of protein release is dependent on a

F. Marffy and M. R. Kula, Biotechnol. Bioeng., 1974, 16,623.

J. R. Woodrow and A. V. Quirk, Enzyme Microb. Technol., 1982,24385.

5 S . T. Harrison, J. S. Dennis, and H. A. Chase, Biosepururion, 1991,2,95.

D. Foster, BiolTechnology, 1992, 10, 1539.



466



Chapter I7



number of factors, including cell type, fermentation conditions, concentration and pre-treatment, such as freezing, as it is often observed that

microbial cells break more easily if they have first been frozen. It has also

been found that the presence of inclusion bodies makes E. coli cells more

easily b r ~ k e n . ~

The rate of protein release from yeast cells can be described by the

empirical first order rate equation:

Log ( R , / R , - R) = K n P"



(1)



Where: R, is the theoretical maximum amount of soluble protein to be

released, R is the actual amount of protein released, K is a temperature

dependent constant, n is the number of passes, P is the operational back

pressure, and a is a constant depending on the organism.

The value of the exponent a varies with the organism; for yeast it was

found to be 2.9.', and for E. coli it is about 2.0. A similar first order

equation can be used to describe the rate of release of proteins from other

organisms, although the value of the exponent varies.' There are many

examples of the use of the Manton-Gaulin homogenizers for the largescale disruption of microbial cells. Beta-galactosidase has been released

from E. coli,'o and carboxypeptidase from Pseudomonas spp." A large

number of enzymes have been isolated from the thermophilic bacterium

Bacillus stearothermophilus, including glycerokinase12 and a glucosespecific he~0kinase.l~

For a reliable process the conditions for cell

breakage must be carefully optimized, as variations in the degree of cell

breakage and protein release can have significant effects on subsequent

purification steps.

3 INITIAL PURIFICATION

3.1 Debris Removal

Following cell disruption, the first step in the purification of an

intracellular enzyme is the removal of cell debris. The separation of

solids from liquids is a key operation in enzyme isolation, and is

normally accomplished by centrifugation or filtration. Many protocols



'A. P. Middelberg, B. K. O'Neill, and D. L. Bogle, Biotechnol. Bioeng., 1991,38,363.



M. Follows, P. J. Hetherington, and M. Lilly, Biotechnol. Bioeng., 1971, 13,549.

C . R. Engler and C. W. Robinson, Biotechnol. Bioeng., 1981,23, 765,

J. J. Higgins, D. S. Lewis, W. Daly, F. G. Mosqueira, P. Dunnill, and M. D. Lilly, Biotechnol.



LO



Bioeng., 1987, 20, 159.

F. Sherwood, R. G. Melton, S. M. Alwan, and P. Hughes, Eur. J. Biochem., 1985,148,447.

12P. M. Hammond, T. Atkinson, and M. D. Scawen, J. Chromatogr., 1986,366,79.

I3C. R. Goward, T. Atkinson, and M. D. Scawen, J. Chromatogr., 1986,369,235.

" R.



Downstream Processing: Protein Extraction and Purijication



467



also include the addition of small quantities of DNase at this point, to

break up long DNA chains which can cause the extract to become

gelatinous.



3.2 Batch Centrifuges

Batch centrifuges are available with capacities ranging from less than

1 ml up to several litres, and capable of applying a relative centrifugal

force of up to 100000 x g (gravitational constant). However, for the

removal of bacterial cells, cell debris and protein precipitates, fields up to

20000 x g are adequate. Many centrifuges of this type, suitable for

intermediate-scale preparations, are available.



3.3 Continuous-flow Centrifugation

Because of the large volumes of liquid which need to be handled at the

beginning of a large-scale enzyme purification, it is preferable to use a

continuous flow centrifuge to remove particulate matter. Three main

types of centrifuge are available; the hollow bowl centrifuge, the disc or

multi-chamber bowl centrifuge, and the basket centrifuge. Hollow bowl

centrifuges have a tubular rotor which provides a long flow path for the

extract, which is pumped in at the bottom and flows upwards through

the bowl. Particulate matter is thrown to the side of the bowl, and the

clarified extract moves up and out of the bowl into a collecting vessel. As

centrifugation proceeds, the effective diameter of the bowl decreases, so

reducing the settling path and the centrifugal force which can be applied.

The ease with which the bowl can be changed, and the possibility of using

a liner to aid sediment recovery has contributed to the popularity of this

type of centrifuge. The flow rate must be determined empirically as it will

vary from one type of extract to another, but a rate of about 60 1 h-' is

generally satisfactory for the larger machines. Centrifuges of this type are

produced by Pennwalt Ltd. (Camberley, Surrey, UK) and by Carl

Padberg GmbH (Lahr, Germany).

Disc centrifuges provide an excellent means of clarifying crude

extracts, and in many cases the sediment may be discharged without

interrupting the centrifugation process. The bowl contains a series of

discs around a central cone. As the extract enters, particulate matter is

thrown outwards, impinging on the coned discs and sedimented matter

collects on the bowl wall. This provides a constant flow path, so there is

little loss of centrifugal efficiency during operation. A disadvantage of

these centrifuges is that some loss of product may be experienced during

the discharge process.



468



Chapter I7



The rotors of instruments which do not have the facility to discharge

sediment during operation are tedious to clean and this again may result

in loss of product if the solids are required. These centrifuges achieve an

RCF (rotational centrifugal force) of about 8000 x g and have a

capacity of up to 20 kg of sediment. As with hollow bowl centrifuges,

the correct flow rate must be determined empirically. A variation of the

disc-type centrifuge is the multi-chambered bowl centrifuge, in which the

bowl is divided by vertically mounted cylinders into a number of

interconnected chambers. The feed passes through each chamber from

the centre outwards, before leaving the centrifuge. This type of arrangement also ensures a short and constant settling path as the bowl fills, and

is easier to dismantle and clean than the disc-type of centrifuge.

Typical centrifuges of these types are produced by De Lava1 Separator

Co. (New York, USA), and by Westfalia Separator Ltd. (Wolverton,

UK).

A problem suffered by all types of centrifugation when applied on an

industrial scale to enzyme recovery is that many homogenates produce

wet sloppy precipitates, which reduces the efficiency of centrifugation. l4

The degree of clarification achieved with an industrial continuous flow

centrifuge is never as great as that obtained with a laboratory centrifuge,

and subsequent steps may be necessary to achieve the desired degree of

clarification. One approach to alleviating this problem is to add a coarse

microgranular cellulose anion exchanger (cell debris remover (CDR),

Whatman) which binds cell debris and increases its density such that it is

removed more efficiently by low speed centrifugation.



3.4 Basket Centrifuges

These are designed to operate at much lower g forces, perhaps only 1000

rev min-I, and are basically centrifugal filters. The bowl is perforated

and is normally lined with a filter cloth. The main use of these centrifuges

is to collect large particulate material; in the context of enzyme purification this usually means ion exchange materials which have been used for

the batch adsorption of the desired protein. Examples of such centrifuges

are available from Carl Padberg GmbH (Lahr, Germany).

3.5 Membrane Filtration

Filtration is an alternative method of clarifying cell extracts. However,

microbial broths and extracts tend to be gelatinous in nature, and are

14



M.Hoate, P.Dunnill, and D. J. Bell, Ann. N . Y.Acad. Sci., 1983,413,254.



Downstream Processing: Protein Extraction and PuriJication



469



difficult to filter by traditional methods, unless very large filter areas are

employed.

This can be overcome by using tangential or cross-flow filtration. In

this method the extract flows at right-angles to the direction of filtration,

and the use of a high flow rate tends to reduce fouling by a self-scouring

action, although this action must be balanced against the possibility of

losses due to shear effects. Membranes with an asymmetric, anisotropic

pore structure are less prone to blockage than isotropic membranes, and

have been used for the large-scale recovery of L-asparaginase from

Erwinia chrysanthemi. A 1 m2 membrane assembly was used to harvest

the cells from 100 litre of culture fluid in 2.5 h, when the solids

concentration in the retentate increased from 0.55% to 22% dry weight.

This same membrane assembly was then used to clarify the extract

obtained by the alkali lysis of these bacteria. These data indicated that to

harvest the cells from 500 litre culture in 2.5 hours would require 7.5 m2

of membrane, and that the costs compared favourably with the costs of

centrifugation. Membranes suitable for cross-flow filtration are available as spirally wound cartridges, which offer the same surface area as

flat membranes, but in a more compact space.

Because of the limitations of large-scale centrifugation the two

techniques are often combined to ensure that the extract is sufficiently

clear for subsequent chromatography.



4 AQUEOUS TWO-PHASE SEPARATION

An alternative to centrifugation or filtration is aqueous two-phase

separation. Aqueous two-phase systems are typically created by mixing

solutions of polyethylene glycol and dextran or polyethylene glycol and

salts such as potassium phosphate or ammonium sulphate to form two

immiscible phases. Proteins and cellular debris show differential solubility between the two phases, so that the technique can be used both for

the separation of proteins from cellular debris and for the partitioning of

enzymes during protein purification. The precise partitioning of a

protein depends on parameters such as its molecular weight and charge,

the concentration and molecular weight of the polymers, the temperature, pH and ionic strength of the mixture and the presence of polyvalent

salts such as phosphate or

The optimal conditions required

"M. S.Le and T. Atkinson, Process Biochem., 1985,20,26.

16H. Walter, D. E. Brooks, and D. Fisher, 'Theory, Methods, Uses and Applications to

Biotechnology: Partitioning in Aqueous Two Phase Systems', Academic Press, New York, 1985.

"N. L. Abbot, D. Blankschtein, and T. A. Hatton, Bioseparation, 1990,1, 191.

F. Tjerneld and G. Johansson, Bioseparation, 1990,1,255.



'*



470



Chapter 17



for a particular protein are found empirically. Although the conditions

required to achieve satisfactory separation can often be precisely defined,

the mechanism of partitioning is not fully understood.''

The phases can be separated in a settling tank, but a more efficient

and rapid separation can usually be achieved by centrifugation. Since

it is easier to separate liquids of different density than solids from

liquids on the large scale, this approach can be used to advantage in

large-scale enzyme purification. Although the relatively low cost of the

polyethylene glycol-salt system makes it attractive for large-scale use,

the more generally useful polyethylene glycol-dextran system can also

have economic advantages in comparison with other purification

methods, despite the high cost of purified dextran, providing the

total processing costs are evaluated.20 Its use is not restricted to

materials of microbial origin, and the method has been successfully

used to isolate materials from both plant2' and

sources,

including human alpha-L-antitrypsin expressed in transgenic milk. In

this case the relatively high starting purity of the protein meant that

after a single two phase separation the desired protein was 73%

pure.23

Aqueous two phase separation can be adapted to offer biospecific

partitioning by attaching ligands to the polymers in order to alter the

partitioning of a

All phase-forming polymers can have

ligands attached covalently to them, and a wide range of such ligands

have been investigated. Because of their simple coupling chemistry, the

reactive dyes have frequently been used as l i g a n d ~ . *The

~ power of the

technique was demonstrated by the %-fold purification of yeast

phosphofructokinase that could be obtained in two steps using Cibacron Blue F-3GA immobilized on polyethylene

Besides the

reactive dyes, a number of other ligands have been investigated. These

include cofactors, such as the pyridine nucleotides used successfully in

the affinity partitioning of a number of dehydrogenases.

On the process scale, affinity partition has been used for the purification of formate dehydrogenase from 10 kg quantities of the yeast

J. Huddleston, A. Veide, K . Kohler, J. Flanagan, S. Enfors, and A. Lyddiatt, Trends Biotechnol.,

1991,9, 381.

2o K . H. Kroner, H. Hustedt, and M. R . Kula, Process Biochem., 1984, 19, 170.

2' H. Vilter, Bioseparation, 1990, 1,283.

22 M . J. Boland, Bioseparation, 1990, 1, 293.

23 D. P. Harris, A. T. Andrews, G. Wright, D. L. Pyle, and J. A. Asenjo, Biosepararion, 1997, 7,31.

24 G. Johansson and F. Tjerneld, in 'Highly Selective Separations in Biotechnology', ed. G. Street,

Blackie Academic and Professional, London, 1994, p.55.

2s G. Kopperschlager, Methods Enzymol., 1994,228, 121.

26G.Johansson, G. Koperschlager, and P. A. Albertsson, Eur. J . Biochem., 1983, 131, 589.

l9



Downstream Processing: Protein Extract ion and Pur ifica t ioli



47 1



Candida bodinii, using the triazine dye, Procion Red HE-3B, immobilized

on polyethylene

Although aqueous two-phase separation is a method which can easily

be scaled up to a manufacturing level, it does not appear to be often used

for industrial-scale purifications.



5 PRECIPITATION



5.1 Ammonium Sulfate

Salting out of proteins has been employed for many years, and fulfils the

dual purposes of purification and concentration. The most commonly

used salt is ammonium sulfate, because of its high solubility, lack of

toxicity towards most enzymes, and low cost.

The precipitation of a protein by salt depends on a number of factors:

pH, temperature, protein concentration, and the salt used.28The protein

concentration is particularly important when scaling-up, because most

large-scale purifications are carried out at higher protein concentrations

than laboratory-scale purifications. This can have a dramatic effect on

the concentration of salt needed to precipitate a given protein.



5.2 Organic Solvents

The addition of organic solvents to aqueous solutions reduces the

solubility of proteins by reducing the dielectric constant of the medium.

Various organic solvents have been used for the precipitation of proteins,

with ethanol, acetone, and propan-2-01 being the most important.

Because proteins are denatured by organic solvents it is necessary to

work at temperatures below 0°C.

Because of their flammable nature, requiring flameproof equipment to

be used, and high cost, coupled with a low selectivity, organic solvents

are not often used in large-scale enzyme purification. The one notable

exception is in the blood processing field, where ethanol precipitation is

the major method for the purification of albumin; indeed it has been

developed into a highly automated, computer-controlled system.29



’’M.R. Kula, in ‘Extraction and Purification of Enzymes. Applied Biochehmistry and Bioengineer-



ing’, ed. L. B. Wingard, E. Katchalski-Katzir, and L. Goldstein, Academic Press, New York,

1979, p.71.

28 M. C. Dixon and E. C. Webb, ‘The Enzymes’, Longmans, London, 1979.

29 P. Foster and J. G. Watt, in ‘Methods of Plasma Fractionation’, ed. J. Curling, Academic Press,

New York, 1980, p.17.



472



Chapter I7



5.3 High Molecular Weight Polymers

Other organic precipitants which can be used for the fractionation of

proteins are water-soluble polymers like polyethylene glycol. This has

the advantage of being non-toxic, non-flammable and not denaturing to

proteins. It is mainly used in the blood processing field.



5.4 Heat Precipitation

When a protein is sufficiently robust, heat treatment can provide a high

degree of purification as an initial step. In a large-scale example, 55% of

unwanted proteins were removed in a single step by heating an E. coli

extract containing recombinant Staphylococcal Protein A at 80" for

10 m h 3 ' If the recombinant protein to be purified is from a thermophilic

organism the results of an initial heat treatment can be even more

dramatic: when an E. coli extract containing recombinant malate

dehydrogenase from Thermus aquaticus is heated to 80" for 20 min the

enzyme in the supernatant is about 90% homogene~us.~'

6 CHROMATOGRAPHY

The purification of proteins by chromatography has been a standard

laboratory practice for many years. These same chromatographic

techniques can equally well be applied to the isolation of much larger

quantities of protein, although the order in which they are used must be

considered carefully.

For the purification of high value/low volume products, typically

therapeutic or diagnostic proteins, chromatography is the most widely

used method. Chromatography is the only method with the required

selectivity to purify a single protein from a complex mixture of proteins

to a final purity of greater than 95%.

6.1 Scale-up and Quality Management

In analytical chromatography, as well as in many laboratory-scale

applications, the quantity of sample to be applied is small, and the

overall aim is to achieve the maximum number of peaks or to produce a

small amount of highly purified protein. The flow rates used are low, as

'OK.A. Philip, P. M. Hammond, and G. W. Jack, Ann. N . Y. Acad. Sci., 1990,613,863.

3 1 R. M. Alldread, D. J. Nicholls, T. K. Sundaram, M.D. Scawen, and T. Atkinson, Gene, 1992,114,

139.



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