RECOVERY / SEPARATION OF PROTEIN FROM ANIMAL TISSUES BY AQUEOUS

TWO PHASE SYSTEM

Gauhar Saddique

Mphil Biotechnology

University of Malakand

DOWN STREAM PROCESSING

integral part of any product development.four stages namely, recovery, isolation,

purification and polishing (RIPP).purification step itself makes up more

than 70% of the total downstream processing costs.

In each step, some quantity of target molecule is lost resulting in a big overall loss.

DOWN STREAM PROCESSING….

conventional techniques for down stream processing such as chromatography, electrophoresis and precipitation have been widely employed.

these methods are considerable cost, providing low yields and not suitable for large scale production

AQUEOUS TWO PHASE SYSTEM

GENERAL ACCOUNT

developed in Sweden during mid-1950s.

initially applied to the separation of plant organelles and viruses.

Now applied in several fields of biotechnology such as recovery of proteins, enzymes, biopharmaceuticals and extractive fermentation

GENERAL ACCOUNT…….

It is a liquid – liquid extraction method which makes use of two aqueous phases.

containing high water content in both liquid phases up to 70-90%.

The two aqueous phases consists of two water-soluble polymers i.e, PEG and polymers like dextran, starch, polyvinylalcohol, etc. or a polymer and a salt i.e, PEG and phosphate or sulphate salts.

PHASE COMPOSITION

1. Polymer / polymer

2. Polymer / Salts

POLYMER / POLYMER PHASE

comprised of PEG and polymers like dextran, starch, polyvinylalcohol, etc.

PEG and dextran system is commonly use, the high cost of fractionated dextran is a limitation for its application in large scale process

POLYMER / SALTS PHASE

composed of PEG and phosphate or sulphate salts.

Potassium dihydrogen phosphate, potassium chloride, sodium dihydrogen phosphate, sodium carbonate, sodium citrate, magnesium sulphate and ammonium sulphate, can be used with PEG.

POLYMER / SALTS PHASE……..

widely used due to several advantages, including: higher selectivity, lower cost, and lower viscosity in biomolecule partitioning in comparison with polymer-polymer systems.

also have a wide application and the range of system pH (from 6 to 9) under which the two phase systems are stable

PHASE PROPERTIES

A number of different chemical and physical interactions are involved in between the two phases, for example hydrogen bond, charge interaction, van der Waals’ forces, hydrophobic interaction and steric effects.

The interfacial tension between the two phases is low, resulting in high mass transfer.

PHASE PROPERTIES….

the distribution of molecules between the two phases depends upon the molecular weight and chemical properties of the polymers and the partitioned molecules of both the phases.

adding solution to the system, mixing and phases settle, the partitioning of target product should be one side whereas the undesirable particles such as cells, cell debris, other proteins and contaminants distribute to the opposite phase

APPLICATIONS

Separation of membrane proteins, for example cholesterol oxidase and bacteriorhodopsin.

For structural analysis of the biological membranes such as thylakoid membranes.

For the concentration and purification of viruses.

APPLICATIONS…….

For bioremediation.

For retroviral vectors purification as an apt substitute for microfiltration, ultra filtration and chromatography protocols.

ADVANTAGES

Substantially reduces the number of initial downstream steps

An ideal extraction technology, specially for proteins, since it is less time consuming and has the potential to give high yield and high purity, involving low investment, less energy, and lower labor costs

ADVANTAGES……

scale-up processes based on aqueous two phase systems are simple, and a continuous steady state is possible.

Straight-forward and requires relatively simple equipments which are easy to operate.

ADVANTAGES….

They provide mild conditions that do not harm or denature unstable/labile biomolecules.

Separation of the phases and the partitioning of the compounds occurs rapidly. This allows the extraction of the desired molecule before endogenous proteases can degrade them.

DISADVANTAGES

. High cost of materials involved, namely high-purity

dextrans employed for the purpose.

However, other low-cost alternatives such as less refined dextrans, hydroxypropyl starch derivatives and high-salt solutions are also available.

SEPARATION OF PROTEIN FROM ANIMAL TISSUE BY ATPS

GENERAL APPROACH

animal tissue generally occurs as a solid mass

To extract the protein(s) of interest invariably requires massive tissue disruption

Further preparation of the protein then necessitates separation from the insoluble particulate material that arises from such disruption

GENERAL APPROACH

The ability to take a crude homogenate, and after a single unit operation deliver a clear solution of partially purified protein, makes this technology in principle an attractive alternative to traditional methods

extraction of a protein from animal tissue will involve coarse disruption, usually by grinding or mincing, followed by fine disruption in the presence of extraction medium, by homogenization

GENERAL APPROACH

Phase-forming components are added to the resulting slurry and phases are separated by centrifugation

The aqueous two-phase system is designed so that the protein(s) of interest will partition into the upper phase, leaving the particulate material in the lower phase

A second phase separation may be induced in the separated upper phase to effect further purification and sometimes to allow recycling of the upper phase components

MATERIALS FOR PROTEIN RECOVERY

Animal TissuePhase Forming Material (PEG)Second Phase Material (Salts)OR Second Phase Material

(Polymers)Affinity Phase MaterialsLaboratory EquipmentsProcess Equipment

1. ANIMAL TISSUE

animal tissue for extraction of enzymes and other animal proteins are usually obtained from an appropriate slaughterhouse or butcher.

Tissue must be as fresh as possible

2. PHASE FORMING MATERIAL

Polyethylene Glycol (PEG):

Most phase forming systems use PEG. This is available from many chemical

suppliers.

3. SECOND PHASE MATERIAL

The second (lower) phase will generally involve a salt such as sodium, potassium or ammonium phosphate, citrate, sulfate, and so forth.

Such salts are generally available from most chemical suppliers

4. SECOND PHASE MATERIALThe most common polymer used for

the lower phase is Dextran, although many alternatives are available .

These polymers are usually much more expensive than salts

a lower rate of use may make them viable alternatives.

5. AFFINITY PHASE MATERIALS

Affinity media can also be used to achieve selective separations of proteins from animal tissue.

Such media have traditionally used triazine dyes bound to a polymer

6. LABORATORY EQUIPMENTLaboratory equipment for aqueous

two-phase system extractions from animal tissue is simple. it needs….

A homogenizer Centrifuge capable of 1000g

7. PROCESS EQUIPMENT

Tissue disruption: using commercial or an industrial meat grinder. Finer disruption is necessary to achieve good extraction in short timescales.

Mixing: In-line mixing is needed to form the phase mixture during processing.

Separation: by a disk stack centrifuge.

METHOD

Selection of phase-forming systemsDisruption of tissue and particle size

reductionEffect of tissue on phase diagramsMixingSeparationSecond phase separationSuitability for further separationsRecycling of phase-forming materials

1. SELECTION OF PHASE-FORMING SYSTEMS

A typical choice for extraction of animal proteins would be a PEG–salt system.

2. DISRUPTION OF TISSUE AND PARTICLE SIZE REDUCTION

Initial disruption can be done by the domestic meat grinder.

Further size reduction and initial extraction is achieved by homogenization at laboratory scale.

the tissue is mixed with an appropriate amount of water or buffer.

3. EFFECT OF TISSUE ON PHASE DIAGRAMS

Phase diagrams are normally developed for a particular two-phase system using model solutions of the specific phase-forming components only.

however, because animal tissue contains considerable levels of protein. A tissue weight that is 20% of the total system is a good starting point.

4. MIXING

Proteins have been shown to equilibrate very rapidly between aqueous phases, and provided mixing is thorough, it need not occur over a long period.

At laboratory scale, 1 min of mixing using a vortex type test tube mixer has been found to be adequate

5. SEPARATION

centrifugation at 500– 1000g for one minute will normally suffice to give a clean separation of the phases.

There will almost always be a layer of insoluble material at the bottom of the tube

layers of insoluble or lipid material may occur at the interface, and at the top of the upper phase.

Phases can be separated by decanting, or transferred using a Pasteur pipette.

6. SECOND PHASE SEPARATION

This will usually be a PEG–salt system if PEG has been used in the first phase separation.

This step has the benefit of removing the protein in question from the PEG phase into an aqueous salt-containing phase that is immediately suitable for further Purification.

7. SUITABILITY FOR FURTHER SEPARATIONSThe solution of the required protein from

the second separation is usually a clear solution, containing a high level of salt.

This can either be desalted using a method such as dialysis or diafiltration prior to further processing, or

the solution can be directly loaded onto a hydrophobic interaction chromatography system

8. RECYCLING OF PHASE-FORMING MATERIALS

Phase-forming chemicals can be a considerable proportion of the cost of an aqueous two-phase system purification and can also incur costs in waste disposal.

It is possible in process scale to recycle the phase-forming components to save these costs