Purification of Proteins

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Biochemistry

Biochemistry	October 6, 2000
Lecture 16: Purification of Proteins Assigned reading in Campbell: None E&S Library Resesrve: Voet & Voet Biochemistry 2nd ed., Ch. 5 for a detailed description. Key Terms:
Specific Activity ELISA Gel Filtration Chromatography Elution Volume Ion Exchange Chromatography	Hydrophobic Chromatography Affinity Chromatography SDS Gel electrophoresis (SDS-PAGE) Isoelectric focusing (IEF)
Take the Review Quiz on Lecture 16 concepts. 10.18.00 Example for Problem Set #7: SDS-PAGE Gel.

Please note: The shaded sections of these revised lecture notes will not be covered this term.
10.13.00

Questions

How to purify.
How to evaluate purity.

Purification of Proteins
Separation is based on different physical/chemical properties of proteins. For example:

Solubility in different salts/organic solvents
Size
Polarity
Charge
Binding to Specific ligands

Specific Activity: The velocity of the enzyme catalyzed reaction (usually V_max) divided by the amount of enzyme. International Units (IU) are mmol/min/mg.

Steps in any purification:

A) Develope a good assay

Enzyme assay (e.g. hydrolysis of an ester to give a colored product)
Recognition by immunoglobulin (not all interesting proteins are enzymes!)

ELISA: Enzyme linked immunosorbant assay

Primary Ab binds to protein of interest
Secondary antibody (anti-primary antibody constant region) binds to primary Ab
Secondary antibody is attached to an enzyme
Presence of the enzyme on the secondary antibody is detected by a color change.

B) Start with a good initial source.

Selection of tissue source High in protein of interest
Low in proteins that may co-purify
Low in proteases that may destroy the protein of interest.
Purification of a subcellular organelle.
Cloning and over-expression of protein in a simpler organism, e.g. E. coli or yeast.

C) Solubilize the enzyme to obtain a homogeneous solution.

Cell Lysis (mechanical breakage of cells)
Detergents (to reduce hydrophobic interactions)
Salts (to reduce electrostatic interactions)

D) Stabilize the enzyme at all purification steps.

Temperature (Most proteins are mor stable at lower (4°C to -85°C) temperatures.)
Protease inhibitors and antimicrobials prevent peptide bond cleavage.
Ligands (Some proteins are more stable with a specific ligand bound.)
Salts (Proteins with highly charged surfaces may be unstable at low ionic strength.)
Metal ions (Some proteins are more stable with bound metal ions.)

E) Separate the components

Precipitation i) Salts
Aggregation at low salt due to unscreened charged-charged interactions
Precipitation at high salt due to change in water structure, e.g. ammonium sulfate precipitation.
ii) Organic Solvents - effects on water structure and hydrophobic interactions

Column chromatography: General Features
In most cases chromatography is performed in long glass tubes filled with a matrix or resin (particle size similar to a fine sand) that is completely immersed in a buffered salt solution. The mixture of proteins is added to the top of this column and buffer is allowed to flow through the column. As the buffer flows through the column the mixture of proteins is drawn down through the column and interacts with the matrix or resin. The actual mode of separation depends on the nature of the resin. In the case of gel filtration the proteins do not interact with the resin. In the case of other forms of chromatography the protein does stick to the resin and must be eluted by reducing the strength of the interaction between the protein and the resin.

Usually several different chromatographic steps are performed. There is no set order, but a typical order might be cation exchange, gel filtration, and then anion exchange.

Examples of Chromatography i) Gel filtration
The matrix contains pores that allow smaller molecules to enter but excludes larger molecules. Thus larger molecules spend less time in the matrix and elute first. The size of the protein can be estimated from the elution time or volume.
If the total column volume is V_t and V_x is the volume occupied by the resin, then the volume available to the solvent is V_o:
V_t = V_x + V_o

The elution volume, V_e, is the volume of solvent required to elute the protein. The molecular weight of the protein can be obtained from the following formula:

V_e/V_o = a*log(MW) + b

Where "a" and "b" are constants obtained by calibration of the gel filtration column. Note that the native molecular weight is obtained, e.g. Hemoglobin would give a measured molecular weight of 64 KDa.

ii) Ion exchange
The protein sticks to these resins by electrostatic interactions. Resins contain either negative (cation exchange resins) or positive (anion exchange resins) charges. In very general terms a protein will stick to a cation exchange resin below its pI and to an anion exchange resin above its pI. However, what really matters is the local charge distribution on the surface of the protein (i.e. patches of residues with similar charges).
Elution of proteins from ion exchange resins involves either a change in pH that results in a change in the charge of the protein or by increasing the salt concentration. The latter method provides additional ions than compete with the protein for binding sites on the resin.

iii) Affinity
The resin contains a specific group (i.e. ligand or antibody) that causes the protein of interest to bind to the resin. In the first example (ligand affinity) elution can be accomplished by the addition of excess ligand. Note that this is analogous to the ion exchange column. In the case of antibody based affinity columns the protein-antibody binding must be weakened by changes of the pH and/or the salt concentration.

iv) Adsorption
Adsorption based on the surface properties of the protein. The most common example is hydrophobic interaction chromatography. In this case the resin is coated with short-chain alkanes that makes it hydrophobic. Proteins will bind to the resin if they have hydrophobic patches on the surface. Elution is usually accomplished by changing the solvent polarity (i.e. addition of alcohol, etc.).

Evaluation of Purity

No increase in specific activity with subsequent purification steps.
Single polypeptide species by: Mass Spectrometry
Gel Filtration/Electrophoresis
Identification of amino-terminal amino acid residue

Electrophoresis
Application of an electric field, E, to a particle with a charge (q) will generate a force resulting in constant velocity (v). The velocity of a charged particle is directly proportional to the strength of the applied field (E).

What happens when an electric field is applied to a solution of charged proteins?

Negatively charged molecules (anions) migrate to the + terminal (anode).
In electrophoresis, "Anions migrate to the anode."
Positively charged molecules (cations) migrate to the - terminal(cathode)
Neutral molecules don't move.

Isoelectric Focusing (IEF)
In this technique a uniform solution of protein is mixed with a special polymer. The properties of this polymer are such that they will form a pH gradient when an electric field is applied across the solution (the various species of the polymer migrate in solution until they reach their isoelectric point). While this pH gradent is being formed the protein molecules also migrate until they reach their isoelectric point. This is a convenient way to obtain the isoelectric point of a protein as well a to purify complex mixtures of proteins.

SDS gel electrophoresis (SDS-PAGE)
Most electrophoretic separations (including IEF) are performed in gel matrices to avoid the problem of convectional mixing. These gels can either be composed of agarose (a carbohydrate polymer from seaweed) or polyacrylamide (a chemically crosslinked polymer). These materials are very similar to Jello in their macroscopic properties.

In addition to preventing thermal mixing, these gels also act as size exclusion media (similar to gel filtration). Therefore, the electrophoretic mobility of proteins in gels will depend on both the size as well as the charge-to-mass ratio. Therefore, to separate proteins according to size it is necessary to give them to same charge-to-mass ratio. This can be accomplished by denaturing the protein in SDS (sodium dodecyl sulfate, shown below).

The SDS binds to most proteins in a uniform manner giving all proteins the same charge-to-mass ratio.

The electrophoretic mobility is defined as the distance migrated in a certain time period. Thus the mobility is directly proportional to the velocity. The effect of the gel matrix is to produce the following dependence of the molecular weight on the mobility.

log(MW) = c*Mobility + d

Where "c" is a proportionality constant that depends on the gel properties. Each gel has a region where the above equation holds and a plot of log(MW) vs. Mobility will be linear. The determination of molecular weight requires the calibration of the SDS gel using proteins of known molecular weight. Note the similarity between the above equation and that used for gel filtration. For example, if hemoglobin were run as a standard, it would result in a band on the gel at a mobility corresponding to MW = 16KDa, i.e. its monomer molecular weight.

Detection of Separated Proteins in SDS-PAGE or IEF

Coomassie blue (blue dye that binds to proteins)
Fluorescamine (fluorescent compound that reacts with proteins)
Autoradiography (Detection of radioactivity in proteins)
Immunoblots (Use of an antibody against the protein to localize it, similar to an ELISA assay).

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Biochemistry

Lecture 16: Purification of Proteins