protein purification

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PROTEIN PURIFICATION:

PROTEIN PURIFICATION

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PROTEINS P T U E R C I H F N I I C Q A U T E I O N

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MIXTURE PROTEIN CENTRIFUGATION CHROMATOGRAPHY ELECTROPHORESIS

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PROTEINS 1. Complex structure 2. Charged molecule 3. Different sizes 4. Affinity to water 5. Nature of binding to different ions – ( solubility )

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1. Complex structure 2. Charged molecule 3. Different sizes 4. Affinity to water 5. Nature of binding to different ions – (solubility ) Affinity chromatography Ion exchange chromatography Size exclusion chromatography Hydrophobic - interaction chromatography Salting in and salting out, solvent fractionation, iso electric precipitation, Ultra centrifugation Electrophoresis Reverse- phase chromatography Size exclusion chromatography

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CHROMATOGRAPHY AFFI NITY GEL-FILTRATION ION EXCHANGE REVERSE PHASE HYDROPHOBIC INTERACTION

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SALTING IN AND SALTING OUT OF PROTEINS

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SALTING IN AND SALTING OUT Principle : Principle is based upon the ionic strength of the salts and concentration of salts. Ionic strength is the measure of concentration and number of electric charges contributed by the salt Solubility of proteins in the solvent changes if there is increase in the ionic strength or the concentration of salt. Points to be remembered : 1. Salts of divalent cations are far more effective than salts of monovalent cations . Ammonium sulphate is largely preferred.

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SALTING IN AND SALTING OUT OF PROTEINS

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What is chromatography ? It is a physical method of separation of substances based on the affinity towards two phases – stationary phase and mobile phase. How is the affinity created ? Affinity is created by the forces of attraction or repulsion between the molecules of the substance and the molecules of the phases. Idea used ? Idea is to make the substances (to be separated) stationary by binding to the stationary phase and mobile by binding to the mobile phase. Method ? First the substances (to be separated) are made stationary by binding to stationary phase, then gradually de – binding from the stationary phase and increasing the binding to mobile phase. This gradual process makes the separation of different proteins from one another . CHROMATOGRAPHY

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AFFINITY CHROMATOGRAPHY Principle : 1 st step : Here the force of attraction is created between proteins and the ligands which are located on the stationary phase. 2 nd step : Here the gradual de – binding is done by changing the buffer composition of the mobile phase. Points to be remembered : 1. Structural motifs Specific interaction between ligand and protein molecules 3. Interaction should be not too strong .

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ION EXCHANGE CHROMATOGRAPHY Principle : 1 st step : Here the force of attraction (ionic forces) is created between charged proteins and the oppositely charged groups on the stationary phase.. 2 nd step : Here the gradual de – binding is done by changing the buffer composition or the salt concentration of the mobile phase. Points to be remembered : 1. Cation and anion exchange chromatography pH of the mobile phase buffer for binding should be in between pKa of the charged protein molecules and pKa of oppositely charged groups on the stationary phase.

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GEL FILTRATION CHROMATOGRAPHY Principle : Here the separation of proteins is done based upon the size and steric hindrance of the protein. This separation is facilitated by using porous stationary phase. These pores help in differential exclusion or inclusion of proteins which means larger proteins exclude first and smaller proteins exclude last. Points to be remembered : 1. No interaction between proteins and stationary phase – so no loss of bio molecular activity of proteins Separation of bio molecules in aqueous systems – gel fitration type Separation of organic polymers in non aqueous systems – gel permeation type

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HYDROPHOBIC INTERACTION CHROMATOGRAPHY Principle : 1 st step : Here the force of attraction ( vanderwall forces ) is created between hydrophobic patches of proteins and the hydrophobic areas of stationary phase. 2 nd step : Here the gradual de – binding is done by lowering the lyotropic salt concentration of the mobile phase. Points to be remembered : 1. Polar mobile phase creates hydrophobic cavities Binding to stationary phase is regulated by the lyotropic salt concentration

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REVERSE PHASE CHROMATOGRAPHY Principle : 1 st step : Here the force of attraction ( vander wall forces) is created between hydrophobic patches of proteins and hydrophobic groups of stationary phase. 2 nd step : Here the gradual de – binding is done by decreasing the polarity of the mobile phase by adding organic solvents in the mobile phase. Points to be remembered : 1. Polar mobile phase creates hydrophobic cavities. Binding of proteins to the stationary phase is regulated by the polarity of the mobile phase.

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ISO ELECTRIC PRECIPITATION pH or pKa is the activity coefficient of a liquid or solid. Principle : Principle is based upon the fact that when pKa of a proteins is equal to pH of the solvent the result is there is no net electric charge on the proteins. This absence of net electric charges causes the absence of repulsive forces between the protein molecules. This helps in precipitation of proteins. The pH at which there is no net electric charge on each protein is called iso electric pH of that particular protein. Change from this iso electric pH generates net electric charge which causes repulsion between proteins. This prevents the precipitation of proteins.

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ISO ELECTRIC PRECIPITATION

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ULTRA CENTRIFUGATION Principle : Principle is based upon the observation that macro molecules like proteins, sediment at high centrifugal fields. But the separation of individual proteins is achieved by creating a density gradient for all the proteins in the sample. This density gradient in the sucrose solution is created by mixing concentrated sucrose solution and water in the decreasing ratio as the tube is filled, so that density of the medium is greatest at the bottom of the tube. Points to be remembered : Sedimentation of each protein down the density gradient depends upon the particle weight, density and shape of the protein. Position of the protein bands can be located optically or by draining off the contents of tube carefully through pinhole in the bottom and analyzing successive small samples or by freezing the tube and cutting it into thin slices for analysis

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ULTRA CENTRIFUGATION

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SOLVENT FRACTIONATION As the name itself suggests that separation of proteins is done by the solvent Principle : Principle is based upon the concept called dielectric constant of the solvent. We have the aqueous solution of proteins , to that water miscible solvents like ethanol or acetone are added. The result is aggregation and precipitation of proteins. This result is because the dielectric constant of ethanol or acetone is lower than that of water. Molecules having higher dielectric constant have greater binding capacity to the charged portions of other molecules, in contrast Molecules having lower dielectric constant have lesser binding capacity to the charged portions of other molecule

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So, when the solvents with lower dielectric constant than water are added to the aqueous solution of proteins, the result is Decrease in the attractive forces between protein molecules and the solvent system Increase in the attractive forces between R groups of the protein. Decreased attraction causes the proteins to precipitate Increased attraction causes the proteins to aggregate.

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ELECTROPHORESIS This method is used to separate proteins based on their molecular weight but the property used is the charged nature of the proteins Principle : Principle is based upon the fact that charged molecules are attracted towards the oppositely charged poles. But the above fact cannot be used to separate proteins to the absolute efficiency , so a gel medium is introduced which acts as a way through which the charged protein molecules move.. The problem with the above solution is that mobility of the protein molecules through the gel is influenced by their different sizes and charges To overcome the above problem, First the proteins in the sample mixture are treated so that all of them unfold into linear polypeptide chain and are coated with negatively charged SDS molecules. From the above solutions we have ruled out charge, shape and size differences among proteins, facilitating the separation purely based on molecular weight

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ELECTROPHORESIS

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Title : Biochemistry edition : 2 nd edition author : Abert L. Lehninger publishers: kalyan publishers www.separations.us.tosohbioscience.com/.../PrinciplesofChromatography http://www.gelifesciences.com/aptrix/upp01077.nsf/content/protein_purification REFFERENCES