Chapter 5: Protein Isolation

Basic Methods for Protein Isolation How do you get your protein? Solubilization, Denaturation, Stabilization Detection Methods Do you have your protein? ELISA, Antibodies, Functional Assays, Mass Spectrometry

Purification Strategies How do you purify your protein? Charge, Size, Polarity, Specificity, Solubility

Considerations for Protein Isolation

Must have sensitive method for detection. Functional assays, antibodies, analytical platforms Select a good source for the protein. a) Rich source of material. b) Bakers yeast (Saccharomyces cerevisiae) c) E. coli Tissue specificity: Brain vs. kidney vs. eye. Chickens, cows, pigs or rats are often used. Molecular cloning techniques: Over-express proteins (bacteria or mammalian cells) Purification strategy Interrelationships of purification steps to isolate the protein in the desired state (functional or not).

Stability: Proteins Can Denature!!!

How can we stabilize the protein through purification steps!
Denaturation is the process by which a protein loses its native or active shape or conformation. H-bonds, ionic interactions, van der Waals interactions, and hydrophobic interactions can be disrupted. Temperature can play a role cold labile heat labile Protect against-Proteases, Inhibitors, Changes in pH Protein can be air-denatured - egg white meringue - absorption to surfaces (glass, plastic) Damaged by oxidation 02 Heavy metals damage proteins -they bind to protein- Cu+, Hg+ Bacterial contamination can destroy the protein

1. Stabilization of Proteins
Methods of Solubilization: Cell Lysis Hypotonic shock. Cells with high salt inside and no salt outside will swell and rupture as water enters cell Digest bacterial outer membranes (Gram-negative) Hen egg white lysozyme digests b(1-4) linkages in (the glycosidic bonds of)polysaccharides. Mechanical breakage blenders, homogenizers French press - high pressure 20,000 lbs/in2 forced through a small hole disrupts cells Ultrasound or sonication disruption. Cryolysis and Freeze/Thaw

1. Stabilization of Proteins

Characteristic
pH Temperature

Effect
Proteins are isolated and kept at a pH at which they are stable/active Proteins are often unstable at high temperatures and so are normally purified at 0oC

Degradative enzymes

Must adjust pH, temperature, inhibitors to inactivate degradative enzymes present in the cells so that they dont degrade the protein of interest

1. Stabilization of Proteins
Characteristic
Adsorption to surfaces

Effect
Proteins are denatured by contact to air-water interfaces or by contact with glass or plastic surfaces so they are kept relatively concentrated All above conditions must be observed and protein must be kept under nitrogen or argon and kept at low temps. (-70 to 196 oC)

Long term storage

2. Purification Strategy
Fractionation procedures or steps to isolate protein based on physical characteristics.

Characteristic Charge

Procedure 1. Ion exchange 2. Electrophoresis 3. Isoelectric focusing

Polarity

1. Adsorption chromatography 2. Paper chromatography 3. Reverse phase chromatography 4. Hydrophobic interaction

2. Purification Strategy
Characteristic Size Procedure 1. Dialysis and ultrafiltration 2. Gel electrophoresis 3. Gel filtration 4. Ultracentrifugation 1. Affinity chromatography 2. Immunopurification 1. Salt precipitation 2. Detergent solubilization

Specificity

Solubility

2. Purification Strategy: Protein Solubility

Protein solubility depends on the concentration of dissolved ions: Ionic strength

Salting in
At low ionic strength, increases in the concentration of dissolved ions leads to an increase in solubility by weakening the interaction between individual protein molecules. Interactions between protein molecules leads to aggregation (i.e. insolubility of proteins.
+ + + + + + + + + + + + -

Salting out

As the ionic strength increases, they out compete the proteins for water molecules and the proteins become less soluble, aggregate, and fall out of solution.

Ionic Strength

1 2 I ci Zi 2
Ci = the molar concentration of the ith species Zi = its ionic charge For monovalent ions, I (the ionic strength) is the same as M (the molar concentration) 1M Na+ ClZi = +1 Na+ Zi = -1 Cl2 2

[1M * (1) ]Na [1M * ( 1) ]Cl 1 1 I 1 2 2

Ionic Strength
For di- or tri-valent ions, where I is different than M. 1M MgCl2 Mg2+ = 1M, and Z = +2 while Cl- = 2M, and Z = -1

[1M * (2) ]Mg [2M * ( 1) ]Cl

2 2

42 3 2

2. Purification Strategy: Salting Out

Use (NH4)2SO4 : it is a Very Soluble salt that does not harm proteins.

Salting Out
a). At low ionic strength, all of the proteins are soluble b). As the ionic strength increases, the least soluble protein precipitates c). At even higher ionic strengths, further proteins precipitate. This process is continued until the desired protein is precipitated. This process not only allows you to obtain the desired protein, it removes many unwanted proteins in the process

Proteins are least soluble when they are neutral, so these salting out experiments are usually carried out at the pI of the protein (i.e. the isoelectric point where pH=pI, and the net charge on the protein is 0)

Chromatography
Analytical methods used to separate molecules. Involves a mobile and a stationary phase.

Mobile phase is what the material to be separated is dissolved in.

Stationary phase is a porous solid matrix which the mobile phase surrounds. Separation occurs because of the differing binding/ interactions each molecule has with both the mobile and stationary phase. Interactions are different depending on the specific method.

Types of Chromatography
Gas - liquid: Mobile phase is gaseous, stationary phase is liquid usually bound to a solid matrix. Liquid - Liquid: Mobile phase is liquid, stationary phase is liquid usually bound to a solid matrix. If separation is based on ionic interaction the method is called Ion Exchange Chromatography.

If separation is based on solubility differences between the phases the method is called Adsorption Chromatography. If the separation is based on size of molecule the method is called Gel Filtration or Size Exclusion. If the separation is based on ligand affinity the method is called Affinity Chromatography.

Ion Exchange Chromatography

A solid matrix with a positive charge, i.e., R+ can bind different anions with different affinities. We can swap one counter ion for another (R+A-) + B- (R+B-) + AR = Resin and exchanges Anions (-) This is an anion exchange resin the stationary phase is decorated with positively charged groups which bind anions in the mobile phase There are also cation exchange resins. The type of an R group can determine the strength of interaction between the matrix, R and the counter ion. If R is R- (R-A+) + B+ (R-B+) + A+

Proteins Are Charged

The charge is positive below pI (more H+) The charge is negative above pI (less H+) The choice of exchange resin depends on the charge of the protein and the pH at which you want to do the purification. Once the protein binds, all unbound proteins are washed off the column. Bound proteins are eluted by increasing the ionic strength, changing the counter ion or changing the pH altering the charge on the protein or the column.

Ion Exchange Chromatography

(a) The mixture of proteins applied to column (purple disc). (b) The salt concentration is low at the beginning, elute proteins with the lowest affinity for the column first (red protein) (c) The salt concentration is then increased, eluting proteins that interact more strongly with the ion exchange column. The most frequently used anion exchanger is: diethylaminoethyl (DEAE) Matrix-CH2-CH2-NH(CH2CH3)2+ The most frequently used cation exchanger is: carboxymethyl (weak) or sulfate (strong) Matrix-CH2-COO-, Matrix-CH2-CH2-SO3 -

Gel Filtration Chromatography

Each gel bead consists of a gel matrix (wavy lines in the brown spheres) Small molecules (red dots) can fit into the internal spaces in the beads and get stuck Larger molecules (blue dots) cannot fit into the internal spaces in the beads and they come through the column faster

Hydrophobic Interaction Chromatography Reversed Phase Chromatography The chromatography column is coated with hydrophobic molecules (butyl (C4), octyl (C8), octadecyl (C18), or phenyl groups) which interact with hydrophobic residues on the surfaces of the proteins. Often performed at low pH (2.5-3.0). Elution is typically increasing hydrophobic content of an aqueous solvent (increasing acetonitrile or methanol in water). Decreasing ionic strength (i.e. lower salt concentration), increasing detergent concentration (which disrupt hydrophobic interactions and lead to unfolding of the proteins), and changes in pH can also affect elution.

Affinity Chromatography
Ligands (yellow in the figure to the left) are attached to the solid resin matrix Proteins in solution have ligand binding sites. However, only one of them will have the binding site for the ligand attached to the solid resin matrix The proteins that do not have the proper ligand binding site will flow through the column fastest The desired protein (i.e. the one with the proper ligand binding site) is then recovered from the column by washing with a solution with high ligand concentration, altered ionic strength, or altered pH

Affinity Chromatography
Based on molecular complementary between an enzyme and substrate. The substrate (R) is linked to a matrix with a spacer arm

Only protein that binds R will stick to column. For example, put citrate on column citrate dehydrogenase will specifically bind. Add excess citrate and the enzyme will be released.

Electrophoresis (PAGE)
Electrophoresis is a method for separating proteins based on how they move in an electric field Image to the left is an electrophoretogram of serum, stained with amido black The sample starts at the top, an electric field is applied, and proteins migrate The molecules at the bottom are the lightest Molecules of similar charge and size move through the gel as a band The pH is typically 9 in these experiments so most proteins have a net negative charge and move toward the positive electrode (i.e. the one attached to the bottom of the gel) Gels are typically made of polyacrylamide and so the experiment is called polyacrylamide gel electrophoresis (PAGE)

SDS-PAGE
Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), SDS-PAGE is used to separate protein mixtures in a protein denaturing environment (SDS soap) That is, the SDS causes proteins to denature and take on a rodlike shape and have similar charge to mass ratios Therefore, proteins are separated by molecular mass Again, the lighter proteins travel further In the figure, several (8) protein mixtures are run at the same time, some are controls and the others are samples Each sample is in a separate column, called a lane

2D Gel Electrophoresis
Combination of isoelectric focusing and SDS-PAGE First isoelectric focusing is carried out in one direction Then subjected to SDS-PAGE in the perpendicular direction

Gel Filtration (Molecular Sieve)

Ultracentrifugation
A centrifuge is an instrument that rotates, generating centripetal forces in excess of 800,000 times that of gravity This causes molecules in solution to undergo sedimentation at different rates, which are related to their masses The rate of sedimentation is measured in s which is the sedimentation velocity per unit of centrifugal force They are normally expressed in units of S (Svedbergs). One Svedberg is 10-13s Proteins: 1-50S Viruses: 40-1000S Organelles: tens of thousands of S

Lysate - broken (lysed) cells- can be separated using differential centrifugation RPM - spun down separates by density differences or by size (MW) of particles. Cellular fractionation can separate mitochondria microsomes ribosomes soluble proteins

Enzyme-linked immunosorbent assay (ELISA)

When proteins are purified, there must be some way of detecting (assaying) for detecting the presence and amount of protein The assay must be performed at each purification step in order to follow protein purification Direct enzyme assay if target is an enzyme Coupled enzymatic assay if protein/enzyme cannot be detected directly Immunoassays are among the most sensitive
Use antibodies raised against the target protein (antigen) A protein mixture containing the protein of interest can be separated since ONLY the antigenic protein will bind to the antibody ELISA is a specialized version of an immunoassay The binding of antigen to antibody is detected by the binding of a second antibody to the antigen that has a coupled enzymatic activity that can be detected (e.g., fluorescence, etc.)

Basics of Detection
Western Blot
Detection by antibody to target protein Dependent on availability and quality of antibodies

ELISA
Enzymatic or antibody-based detection Amenable to antibody-based approaches Dependent on availability and quality of antibodies

Mass Spectrometry
Direct coupling with antibody-based approaches No antibodies are necessary, but helpful Capable of ID for unexpected proteins