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Lecture 7: Protein purification

–Protein purification

Methods of solubilization

•1st step is to get the protein into solution

•If the protein is located in the cytosol, we need to break open the cell.

•For animal cells this can be accomplished with osmotic lysis. Put the cellsin hypotonic solution; less solutes than inside the cell.

•For cells with a cell wall (bacteria, plants) we have to use other methods.

•For bacteria, lysozyme is often effective - Selectively degrades bacterialcell wall.

•Can also use detergents or organic solvents, although these may alsodenature the protein.

Mechanical processes to break open thecell

•High speed blender

•Homogenzier

•French press

•Sonicator.

After the cells have been broken, the crude lysate, may be filtered orcentrifuged to remove the particulate cell debris. The protein of interest isin the supernatant.

For proteins that are components ofmembranes or subcellular assembly

•Remove the assembly from the rest of the cellular material (mitochondriafor example).

•Can be done by differential centrifugation-cell lysate is centrifuged at aspeed that removes only the cell components denser than the desiredorganelle followed by a centrifugation at speed that spins down theorganelle.

Stabilization of proteins

•pH

•Temperature

•Inhibition of proteases

•Retardation of microbes that can destroy proteins

–Sodium azide is often used.

Assay of proteins

•Done throughout the purification process to make sure that your protein ofinterest is there.

•If the protein of interest is an enzyme, using a reaction for which thatenzyme is a catalyst is a good way to monitor protein recovery.

•Monitor the increase of the product of the enzymatic reaction

–Fluorescence

–Generation of acid to be monitored by titration

–Coupled enzymatic reaction - couple with another enzyme to make anobservable substance.

Immunochemical techniques to assay forproteins

•Use specific immunoglobulins (antibodies), proteins that interactspecifically with the protein of interest and can be easily monitored.

•Antibodies are produced by an animal’s immune system in response to theintroduction of a foreign protein.

•Antibodies specifically bind to the foreign protein.

•Extracted from blood serum of animal that has been immunized against aparticular protein.

•Many different antibodies in sera with different specificities and bindingaffinities toward the protein of interest.

Immunochemical techniques to assay forproteins

•Immune cells that produce antibodies normally die after a few cell divisionsso it is difficult to get a specific antibody clone.

•We can make monoclonal antibodies by fusing a cell producing thedesired antibody with a cancer cell (myeloma).

–This results in a hybridoma that is essentially an immortal cell, so largequantities of monoclonal antibody can be produced.

Figure 6-1An enzyme-linked immunosorbentassay (ELISA).

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Summary of initial steps of proteinpurification

•Choose source of proteins.

•Solubilize proteins.

•Stabilize proteins.

•Specific assay for protein of interest

–Enzymatic activity, immunological activity, physical characteristics (e.g.molecular mass, spectroscopic properties, etc.), biological activity

•Assay should be:

–Specific

–Rapid

–Sensitive

–Quantitative

Things to monitor during proteinpurification

•Things to monitor during protein purification

–Total sample volume

–Total sample protein (est. by A280; 1.4-1.0 mg/ml)

–Units of activity of desired protein (based on specific assay)

•Other basic information to track

–% yield for each purification step

–Specific activity of the desired protein (units/mg of protein)

–Purification enhancement of each step (e.g. “3.5 fold purification”)

•In designing a purification scheme you have to balance purificationwith yield.

Characteristic

Procedure

Solubility:

1.Salting in

2.Salting out

Ionic charge:

1.Ion exchange chromatography

2.Electrophoresis

3.Isoelectric focusing

Polarity:

1.Adsorption chromatography

2.Paper chromatography

3.Reverse-phasechromatography

4.Hydrophobic interactionchromatography

Molecular size:

1.Dialysis

2.Gel electrophoresis

3.Gel filtration chromatography

4.Ultracentrifugation

Binding specificity:

1.Affinity chromatography

Solubilities of proteins

•Multiple acid-base groups on proteins affect their solubility properties.

•Solubility of a protein is therefore dependent on concentrations of dissolvedsalts, the polarity of solvent, the pH and the temperature.

•Certain proteins will precipitate from solutions under conditions which othersremain soluble-so we can use this as an initial purification step of proteins.

•Salting out or salting in procedures take advantage of ionic strength

1/2ciZi

Ionic strength (I) =

Ci = molar concentration of ionic species

Zi = ionic charge

Figure 6-2Solubilities of several proteins inammonium sulfate solutions.

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Solubilities of proteins

•A protein’s solubility at a given ionic strength varies with the types of ions in solution.

•The order of effectiveness of these various ions in influencing protein solubility isquite similar for different proteins and is due to the ion’s size and hydration.

•The solubility of a protein at low ionic strength generally increases with the saltconcentration. This is called salting in. As the salt concentration increases theadditional counterions more effectively shield the protein molecule’s multiple ioniccharges and thereby increase the protein’s solubility.

•At high ionic strengths the solubilities of proteins as well as most other substancesdecrease. This is called salting out and results from a competition between theadded salt ions and the other dissolved solutes for molecules of solvation.

Figure 6-3Solubility of caboxy-hemoglobin atits isoelectric point as a function of ionicstrength and ion type.

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Ammonium Sulfate(% saturated)

Sample A280

1000

900

600

300

100

Activity assay (units)

200

190

170

100

Solubilities of proteins

•Salting out is one of the most commonly used proteinpurification procedures.

•By adjusting the salt concentration in a solution with a mixture ofproteins to just below the precipitation point of the protein to bepurified, many unwanted proteins can be eliminated from solution.Then after the precipitate is removed by filtration or centrifugation,the salt concentration of the remaining solution is increased toprecipitate the desired protein.

•Ammonium sulfate is the most commonly used reagent

–High solubility (3.9 M in water at 0 ºC)

–High ionic strength solution can be made (up to 23.5 in water at 0 ºC)

Note-certain ions (I-, ClO4-, SCN-, Li+, Mg2+, Ca2+ and Ba+) increase thesolubilities of proteins rather than salting out. (also denature proteins).

Solubilities of proteins

•Water-miscible organic solvents also precipitateproteins.

–Acetone, ethanol

–Low dielectric constants lower the solvating power oftheir aqueouse solutions for dissolved ions.

•This technique is done at low temperatures (0 ºC)because at higher temperatures, the solvent evaporates.

•Can magnify the differences in salting out procedures.

•Some water-miscible organic solvents (DMF, DMSO) aregood at solubilizing proteins (high dielectric constants).

Solubilities of proteins

•Proteins have various ionizable groups (many pK’s)

•At a pH characteristic for each protein, the positivecharges on the molecule exactly balance the negativecharges (isoelectric point, pI).

•At pI, the protein has no net charge and is immobile inan electric field.

•Therefore, solubility can be influenced by changes in thepH.

Figure 6-4Solubility of -lactoglobin as afunction of pH at several NaCl concentrations.

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Solubilities of proteins

•A protein in a pH near its isolectric point is not subject tosalting in.

•As the pH is moved away from the pI of the protein, theprotein’s net charge increases and it is easier to salt in.

•Salts inhibit interactions between neighboring moleculesin the protein that promote aggregation and precipitation.

•pI’s of proteins can be used to precipitate proteins.

Table 6-1Isoelectric Points of SeveralCommon Proteins.

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Column chromatography

•After the initial fractionation steps we move to column chromatography.

•The mixture of substances (proteins) to be fractionated is dissolved in aliquid or gaseous fluid called the mobile phase.

•This solution is passed through a column consisting of a porous solid matrixcalled the stationary phase. These are sometimes called resins whenused in liquid chromatography.

•The stationary phase has certain physical and chemical characteristics thatallow it to interact in various ways with different proteins.

•Common types of chromatographic stationary phases

–Ion exchange

–Hydrophobic

–Gel filtration

–Affinity

Ion exchange chromatography

•Ion exchange resins contain charged groups.

•If these groups are acidic in nature they interact with positively chargedproteins and are called cation exchangers.

•If these groups are basic in nature, they interact with negatively chargedmolecules and are called anion exchangers.

DEAE cellulose

anion exchanger

CH2-CH2 -NH+(CH2CH2)

Negativelycharged (acidic)protein or enzyme

CM cellulose

cation exchanger

CH2-COO-

Positivelycharged (basic)protein or enzyme

CH2-CH2 -NH+(CH2CH2)

Ion exchange chromatography

CM cellulose

cation exchanger

CH2-COO-

Positively charged proteinor enzyme bind to thecolumn

For protein binding, the pH is fixed (usually near neutral) under low saltconditions. Example cation exchange column…

Negativelycharged proteinspass through thecolumn

Ion exchange chromatography

CM cellulose

cation exchanger

CH2-COO-

To elute our protein of interest, add increasingly higher amount of salt(increase the ionic strength). Na+ will interact with the cation resin and Cl-will interact with our positively charged protein to elute off the column.

CM cellulose

cation exchanger

CH2-COO-

Na+

Cl-

Na+2

Cl-

Na+2

+ Increasing[NaCl] of theelution buffer

Ion exchange chromatography

•Proteins will bind to an ion exchanger with different affinities.

•As the column is washed with buffer, those proteins relatively low affinitiesfor the ion exchange resin will move through the column faster than theproteins that bind to the column.

•The greater the binding affinity of a protein for the ion exchange column, themore it will be slowed in eluting off the column.

•Proteins can be eluted by changing the elution buffer to one with a highersalt concentration and/or a different pH (stepwise elution or gradientelution).

•Cation exchangers bind to proteins with positive charges.

•Anion exchangers bind to proteins with negative charges.

Figure 6-6Ion exchange chromatographyusing stepwise elution.

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Ion exchange chromatography

•Gradient elution can improve the washing of ion exchange columns.

•The salt concentration and/or pH is continuously varied as the column iseluted so as to release sequentially the proteins bound to the column.

•The most widely used gradient is the linear gradient where theconcentration of eluant solution varies linearly with the volume of thesolution passed.

•The solute concentration, c, is expressed as

c = c2 - (c2 - c1)f

c1 = the initial concentration of the solution in the mixing chamber

c2 = the concentration of the reservoir chamber

f = the remaining fraction of the combined volumes of the solutions initiallypresent in both reservoirs.

Figure 6-7Device for generating a linearconcentration gradient.

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c = c2 - (c2 - c1)f

Figure 6-8Molecular formulas of cellulose-based ion exchangers.

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Table 6-2Some Biochemically Useful IonExchangers.

Ion exchange chromatography

•Ion exchangers can be cellulosic ion exchangers andgel-type ion exchangers.

•Cellulosic ion exchangers most common.

•Gel-type ion exchangers can combine with gel filtrationproperties and have higher capacity.

•Disadvantage-these materials are easily compressed soeluant flow is low.

•There are other materials derived from silica or coatedglass beads that address this problem.

Gel filtration chromatography

•Also called size exclusion chromatography ormolecular sieve chromatography.

How does it work? If we assume proteins are spherical…

size

Molecular mass

(daltons)

10,000

30,000

100,000

Gel filtration chromatography

flow

Gel filtration chromatography

flow

Gel filtration chromatography

flow

Gel filtration chromatography

flow

Gel filtration chromatography

flow

Gel filtration chromatography

•The molecular mass of the smallest molecule unable to penetratethe pores of the gel is at the exclusion limit.

•The exclusion limit is a function of molecular shape, since elongatedmolecules are less likely to penetrate a gel pore than other shapes.

•Behavior of the molecule on the gel can be quantitativelycharacterized.

Total bed volume of the column

Vt = Vx + V0

Vx = volume occupied by gel beads

V0 = volume of solvent space surrounding gel; Typically 35%

Gel filtration chromatography

•Elution volume (Ve) is the volume of a solvent required to elute agiven solute from the column after it has first contacted the gel.

•Relative elution volume (Ve/V0) is the behavior of a particularsolute on a given gel that is independent of the size of the column.

•This effectually means that molecules with molecular massesranging below the exclusion limit of a gel will elute from a gel in theorder of their molecular masses with the largest eluting first.

Figure 6-9Gel filtration chromatography.

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Figure 6-10Molecular mass determinationby gel filtration chromatography.

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Table 6-3Some Commonly Used GelFiltration Materials.

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Gel filtration chromatography

•Elution volume (Ve) is the volume of a solvent required to elute agiven solute from the column after it has first contacted the gel.

•Relative elution volume (Ve/V0) is the behavior of a particularsolute on a given gel that is independent of the size of the column.

•This effectually means that molecules with molecular massesranging below the exclusion limit of a gel will elute from a gel in theorder of their molecular masses with the largest eluting first.