1. Lecture 7: Protein purification

2. 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 cells in 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 bacterial cell wall.
Can also use detergents or organic solvents, although these may also denature the protein.

3. Mechanical processes to break open the cell
High speed blender
Homogenzier
French press
Sonicator.
After the cells have been broken, the crude lysate, may be filtered or centrifuged to remove the particulate cell debris. The protein of interest is in the supernatant.

4. For proteins that are components of membranes or subcellular assembly
Remove the assembly from the rest of the cellular material (mitochondria for example).
Can be done by differential centrifugation-cell lysate is centrifuged at a speed that removes only the cell components denser than the desired organelle followed by a centrifugation at speed that spins down the organelle.

5. Stabilization of proteinspH
Temperature
Inhibition of proteases
Retardation of microbes that can destroy proteins
Sodium azide is often used.

6. Assay of proteins
Done throughout the purification process to make sure that your protein of interest is there.
If the protein of interest is an enzyme, using a reaction for which that enzyme 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 an observable substance.

7. Immunochemical techniques to assay for proteins
Use specific immunoglobulins (antibodies), proteins that interact specifically with the protein of interest and can be easily monitored.
Antibodies are produced by an animal’s immune system in response to the introduction of a foreign protein.
Antibodies specifically bind to the foreign protein.
Extracted from blood serum of animal that has been immunized against a particular protein.
Many different antibodies in sera with different specificities and binding affinities toward the protein of interest.

8. Immunochemical techniques to assay for proteins
Immune cells that produce antibodies normally die after a few cell divisions so it is difficult to get a specific antibody clone.
We can make monoclonal antibodies by fusing a cell producing the desired antibody with a cancer cell (myeloma).
This results in a hybridoma that is essentially an immortal cell, so large quantities of monoclonal antibody can be produced.

10. Figure 6-1 An enzyme-linked immunosorbent assay (ELISA).
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11. Summary of initial steps of protein purification
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

12. Things to monitor during protein purification
Total sample volume
Total sample protein (est. by A280; 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 purification with yield.
It may be straightforward to get 90% purity but difficult to purify further.
The planned application of the purified product is important. If it is for clinical trials, maybe need 99.99%

13. General flowchart for protein purification.

14. Characteristic
Procedure
Solubility:
Salting in
Salting out
Ionic charge:
Ion exchange chromatography
Electrophoresis
Isoelectric focusing
Polarity:
Adsorption chromatography
Paper chromatography
Reverse-phase chromatography
Hydrophobic interaction chromatography
Molecular size:
Dialysis
Gel electrophoresis
Gel filtration chromatography
Ultracentrifugation
Binding specificity:
Affinity chromatography

15. Solubilities of proteins
Multiple acid-base groups on proteins affect their solubility properties.
Solubility of a protein is therefore dependent on concentrations of dissolved salts, the polarity of solvent, the pH and the temperature.
Certain proteins will precipitate from solutions under conditions which others remain soluble-so we can use this as an initial purification step of proteins.
Salting out or salting in procedures take advantage of ionic strength 2
1/2ciZi
Ionic strength (I) =
Ci = molar concentration of ionic species
Zi = ionic charge

16. Figure 6-2 Solubilities of several proteins in ammonium sulfate solutions.
Solubility of the protein goes down as ionic strength increases for various protein species.
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17. 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 is quite 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 salt concentration. This is called salting in. As the salt concentration increases the additional counterions more effectively shield the protein molecule’s multiple ionic charges and thereby increase the protein’s solubility.
At high ionic strengths the solubilities of proteins as well as most other substances decrease. This is called salting out and results from a competition between the added salt ions and the other dissolved solutes for molecules of solvation.

18. Figure 6-3 Solubility of caboxy-hemoglobin at its isoelectric point as a function of ionic strength and ion type.
Here S and S’ are the solubilities of the protein in the salt solution and pure water.
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 is quite 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 salt concentration. This is called salting in. As the salt concentration increases the additional counterions more effectively shield the protein molecule’s multiple ionnic charges and thereby increase the prortein’s solubility.
At high ionic strengths the solubilities of proteins as well as most other substances decrease. This is called salting out and results from a competition between the added salt ions and the other dissolved soluties for molecules of solvation.
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19. 2. Centrifuge and discard the pellet
Ammonium Sulfate (% saturated) 10 20 30 40 50 60 70 80 90
Sample A280
1000
900
600
300
100 75 25
Activity assay (units)
200
190
170 5
* In this particular example we are in luck: at around 30% ammonium sulfate we can precipitate about 80% of the total protein concentration in our sample, yet our activity assay for our desired protein indicates that about 95% of our desired protein is still soluble.
* At 80% ammonium sulfate all of our desired protein has precipitated. Thus, from these results we would do the following:
1. Add ammonium sulfate to our sample to a concentration of 30% saturation
2. Centrifuge and discard the pellet
3. Add ammonium sulfate to 80% saturation
4. Centrifuge and keep the pellet. Resuspend the pellet in buffer to solubilize the protein.
* We would expect about a 5-fold purification with about 95% yield.

20. Solubilities of proteins
Salting out is one of the most commonly used protein purification procedures.
By adjusting the salt concentration in a solution with a mixture of proteins to just below the precipitation point of the protein to be purified, 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 to precipitate 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 the solubilities of proteins rather than salting out. (also denature proteins).

21. Solubilities of proteins
Water-miscible organic solvents also precipitate proteins.
Acetone, ethanol
Low dielectric constants lower the solvating power of their 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) are good at solubilizing proteins (high dielectric constants).

22. Solubilities of proteins
Proteins have various ionizable groups (many pK’s)
At a pH characteristic for each protein, the positive charges on the molecule exactly balance the negative charges (isoelectric point, pI).
At pI, the protein has no net charge and is immobile in an electric field.
Therefore, solubility can be influenced by changes in the pH.

23. Figure 6-4 Solubility of b-lactoglobin as a function of pH at several NaCl concentrations.
The solubility of the lactoglobin protein is worst near the pI of beta lactoglobin is We see here that in dilute NaCl solutions the solubility changes based on the pH.
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24. Solubilities of proteins
A protein in a pH near its isolectric point is not subject to salting in.
As the pH is moved away from the pI of the protein, the protein’s net charge increases and it is easier to salt in.
Salts inhibit interactions between neighboring molecules in the protein that promote aggregation and precipitation.
pI’s of proteins can be used to precipitate proteins.

25. Table 6-1 Isoelectric Points of Several Common Proteins.
If you adjust the pH of the protein mixture to the pI of the protein of interest, you can use it with salting out so that the protein being purified is usually salted near its pI.
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26. Column chromatography
After the initial fractionation steps we move to column chromatography.
The mixture of substances (proteins) to be fractionated is dissolved in a liquid or gaseous fluid called the mobile phase.
This solution is passed through a column consisting of a porous solid matrix called the stationary phase. These are sometimes called resins when used in liquid chromatography.
The stationary phase has certain physical and chemical characteristics that allow it to interact in various ways with different proteins.
Common types of chromatographic stationary phases
Ion exchange
Hydrophobic
Gel filtration
Affinity

27. Ion exchange chromatography
Ion exchange resins contain charged groups.
If these groups are acidic in nature they interact with positively charged proteins and are called cation exchangers.
If these groups are basic in nature, they interact with negatively charged molecules and are called anion exchangers.
Positively charged (basic) protein or enzyme
CH2-COO- + +
CH2-COO- + +
CM cellulose
cation exchanger -
Negatively charged (acidic) protein or enzyme
CH2-CH2 -NH+(CH2CH2)
CH2-CH2 -NH+(CH2CH2)
DEAE cellulose
anion exchanger

28. Ion exchange chromatography
For protein binding, the pH is fixed (usually near neutral) under low salt conditions. Example cation exchange column…
Positively charged protein or enzyme bind to the column +
CM cellulose
cation exchanger
CH2-COO- + + + - -
Negatively charged proteins pass through the column

29. Ion exchange chromatography
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-
+ Increasing [NaCl] of the elution buffer
Cl-
CM cellulose
cation exchanger
CH2-COO-
Na+
Na+2 +
Cl-
Na+
Cl-
Na+2
Cl-

30. Ion exchange chromatography
Proteins will bind to an ion exchanger with different affinities.
As the column is washed with buffer, those proteins relatively low affinities for the ion exchange resin will move through the column faster than the proteins that bind to the column.
The greater the binding affinity of a protein for the ion exchange column, the more it will be slowed in eluting off the column.
Proteins can be eluted by changing the elution buffer to one with a higher salt concentration and/or a different pH (stepwise elution or gradient elution).
Cation exchangers bind to proteins with positive charges.
Anion exchangers bind to proteins with negative charges.

31. Figure 6-6 Ion exchange chromatography using stepwise elution.
The tan region of the column represents the ion exchannger and colored bands various proteins.
The protein mixture is bound to the topmost portionof the ion exchanger in the chromatography column.
as the elution progresses the varouse proteins separate into discrete bands as a consequence of their different affinities for the ion exchanger under the prevailing solution conditions. Here the first band of protein (red) has passed through the column and is being isolated as a separate fraction, whereas the other, less mobile, bands remain near the top of the column.
the salt concentration in the elution buffer i9s increased to increase the mobility of and thus elute the remaining bands.
The elution diagramm of the protein mixture.
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32. 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 is eluted so as to release sequentially the proteins bound to the column.
The most widely used gradient is the linear gradient where the concentration of eluant solution varies linearly with the volume of the solution 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 initially present in both reservoirs.

33. Figure 6-7 Device for generating a linear concentration gradient.
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c = c2 - (c2 - c1)f

34. Figure 6-8 Molecular formulas of cellulose-based ion exchangers.
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35. Table 6-2 Some Biochemically Useful Ion Exchangers.

36. Ion exchange chromatography
Ion exchangers can be cellulosic ion exchangers and gel-type ion exchangers.
Cellulosic ion exchangers most common.
Gel-type ion exchangers can combine with gel filtration properties and have higher capacity.
Disadvantage-these materials are easily compressed so eluant flow is low.
There are other materials derived from silica or coated glass beads that address this problem.

37. Gel filtration chromatography
Also called size exclusion chromatography or molecular sieve chromatography.
How does it work? If we assume proteins are spherical…
size
Molecular mass
(daltons)
10,000
30,000
100,000
The gel bead has molecular size holes so that small molecules like water and buffer enter them completely.

38. Gel filtration chromatography
flow
Some proteins are small enough to also enter the molecular holes of the gel bead. Other proteins are too large to enter the holes and pass by the gel bead. The concept is of Reverse Sieve, since a normal sieve retains large and passes small particles. In gel filtration the larger proteins elute first, medim sized ones next and fijnally the smallest elute last.

39. Gel filtration chromatography
flow

40. Gel filtration chromatography
flow

41. Gel filtration chromatography
flow

42. Gel filtration chromatography
flow

43. Gel filtration chromatography
The molecular mass of the smallest molecule unable to penetrate the pores of the gel is at the exclusion limit.
The exclusion limit is a function of molecular shape, since elongated molecules are less likely to penetrate a gel pore than other shapes.
Behavior of the molecule on the gel can be quantitatively characterized.
Total bed volume of the column
Vt = Vx + V0
Vx = volume occupied by gel beads
V0 = volume of solvent space surrounding gel; Typically 35%

44. Gel filtration chromatography
Elution volume (Ve) is the volume of a solvent required to elute a given solute from the column after it has first contacted the gel.
Relative elution volume (Ve/V0) is the behavior of a particular solute on a given gel that is independent of the size of the column.
This effectually means that molecules with molecular masses ranging below the exclusion limit of a gel will elute from a gel in the order of their molecular masses with the largest eluting first.

45. Figure 6-9 Gel filtration chromatography.
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46. Figure 6-10 Molecular mass determination by gel filtration chromatography.
Relative elution volume vs. log of molecular mass for variety of proteins.
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47. Table 6-3 Some Commonly Used Gel Filtration Materials.
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48. Gel filtration chromatography
Elution volume (Ve) is the volume of a solvent required to elute a given solute from the column after it has first contacted the gel.
Relative elution volume (Ve/V0) is the behavior of a particular solute on a given gel that is independent of the size of the column.
This effectually means that molecules with molecular masses ranging below the exclusion limit of a gel will elute from a gel in the order of their molecular masses with the largest eluting first.

49. Affinity chromatography
Many proteins can bind specific molecules very tightly but noncovalently.
We can use this to our advantage with affinity chromatography.
Glucose (small dark blue molecule) binding to hexokinase.
The enzyme acts like a jaw and clamps down on the substrate (glucose)

50. Affinity chromatography
How does it work?
Ligand - a molecule that specifically binds to the protein of interest.
Inert support + +
Spacer arms
Ligand
Inert support
Affinity material
prepared

51. Affinity chromatography
Inert support
Mixture of proteins
Inert support
Unwanted proteins

52. Affinity chromatography
Inert support
Elute with competitive ligand.
Inert support
Remove from competitive ligand
by dialysis.

53. Affinity chromatography
To remove the protein of interest from the column, you can elute with a solution of a compound with higher affinity than the ligand (competitive)
You can change the pH, ionic strength and/or temperature so that the protein-ligand complex is no longer stable.

54. Immunoaffinity chromatography
Monoclonal antibodies can be attached to the column material.
The column only binds the protein against which the antibody has been raised.
10,000-fold purification in a single step!
Disadvantges
Difficult to produce monoclonal antibodies (expensive $$!)
Harsh conditions to elute the bound protein