Protein Purification

What is the table below and where have you seen it before?

Shafqat et al. Eur. J. Biochem. 263, 305−311 (1999)

Overall Strategy

Molecular characteristics of target protein

Look for the properties that distinguish the target from other proteins

Target features

charge/hydrophobicitysolubility & stabilizationsize

Define a way of identifying the presence of your protein, and of assessing its purity, before you start purification

Protein Purification

I. Source II. Method of lysing the cellsIII. Protecting the protein from degradationIV. Means to keep track of your protein – activity assay, protein purification table V. Early Stages – gentle fractionation steps to handle largish volumes.VI. Middle Stages – chromatographic separation(s)VII. Final (Polishing) Stages

Reading: N & B Chapters 6 & 7

I. Source

What source will you use to obtain the protein of interest?

A) Natural – some plant or animal tissue

B) Recombinant – a gene for the protein of interest that has been cloned into a vector and transformed into expression cells

Structural Integrity

“native” versus “non-native” conformation

denaturation/renaturation

protein folding/re-folding subunit dissociation/re-association

Some other considerations

The pKa of the buffer should be within 0.5 unit of the desired pH

Potential interactions with a column matrix of any resins you might use

Avoid UV-absorbing buffers if you plan to use a UV detector

The ionic strength and salt composition must be chosen according to the stability of the protein and the detergent

Choice of Buffer

Buffer

Salt

Detergents (sometimes)

Glycerol, sugars

Metal chelators

Protease inhibitors (usually)

Reducing agents (often)

Ligands (sometimes)

Typical Solution Components

Centrifuge

Be very careful using the centrifuge. Samples mustbe balanced. Clean up any spills, especially thosethat have ammonium sulfate, which is very corrosive.

On December 16, 1998, milk samples were running in a Beckman L2-65B ultracentrifuge using a large aluminum rotor (a rotor is a large metal object that holds the individual sample tubes and is connected to the spin drive of the centrifuge). The rotor had been used for this procedure many times before. Approximately one hour into the operation, the rotor failed due to excessive mechanical stress caused by the "G" forces of the high rotation speed. The subsequent explosion completely destroyed the centrifuge (Images 1 & 2). The safety shielding in the unit did not contain all the metal fragments. The half-inch thick sliding steel door on top of the unit buckled allowing fragments, including the steel rotor top, to escape (Image 3). Fragments ruined a nearby refrigerator and an ultra-cold freezer in addition to making holes in the walls and ceiling. The unit itself was propelled sideways and damaged cabinets and shelving that contained over a hundred containers of chemicals (Image 4). Fortunately, sliding cabinet doors prevented the containers from falling to the floor and breaking. A shock wave from the accident shattered all four windows in the room. The shock wave also destroyed the control system for an incubator and shook an interior wall causing shelving on the wall to collapse (Image 5). Fortunately the room was not occupied at the time and there were no personal injuries.

We will have more to say about centrifugation, especiallyanalytical ultracentrifugation, during a later lecture

II. Method of lysing the cells

How will you “break open” the cells of the tissue or the bacterial cells? Not a trivial matter.

A) Mechanical disruption – homogenizing the tissue with a Waring blendor (note the spelling).

B) French Press

C) Sonication

D) Freeze Thaw Cycles

E) Enzymatic – e.g., Lysozyme or Chemical Methods

http://extranet.libraries.psu.edu/psul/waring/blendor.html

Frederick Waring and his “blendor”

http://upload.wikimedia.org/wikipedia/commons/9/91/French_press.jpg

A French Press, but not the one we are interested in

Analytical Biochemistry 321 (2003) 276–277

This is the French Press we are interested in

http://en.wikipedia.org/wiki/Sonication

http://www.fishersci.com/wps/portal/PRODUCTDETAIL?aid=2819374

Some examples of sonicators

III. Protecting the protein from degradation

OK, so you have figure out a way or method to break openthe cells, now how are you going to protect your proteinfrom degradation?

A) Add PMSF (phenylmethylsulfonylfluoride) to inhibit proteases

B) Add other protease inhibitors

C) High or low pH to inhibit proteases, working quickly

D) Use “Expression” cells

IV. Means to keep track of your protein – activity assay, protein purification table

How are you going to know where your protein is? Is it in the pellet or the supernatant following a centrifugation step?

A) Activity assay – a beacon in the “soup”. Activity versus Specific Activity.

B) At each step you save a sample and later construct a protein purification table.

C) Use of PAGE – subject for later, but not ideal as it is too time consuming.

A41

0

N&B Fig. 7-1

Activity Assay

"Specific Activity"

Definition:Units of enzyme activity per mg protein

1 Unit = amount of enzyme that will convert one µmole of substrate to product in one minute at a given pH (optimum value) and temperature (usually 25°C or 37°C).

Specific activity is used as an estimate of enzyme purity.

You have total of 5 mL of 2 mg/mL protein. From a 3 mL assay containing 1.5 mL of 50 mM substrate and 100 μL of your protein you obtain ∆A/∆t = 0.1 min−1. Calculate the specific activity of the enzyme in the sample. Assume that the substrate has the following extinction coefficient: ε410 nm = 18.8 mM−1 cm−1. Report your answer to one significant figure.

Example

Activity vs. Specific Activity

Purification Table (example) (see N&B p. 172 for more details)

V. Early Stages – gentle fractionation steps to handle largish volumes.

How can I gently separate my protein from a number ofother contaminants using relatively gentle methods?

A) Salting in/Salting out – the use of ammonium sulfate to gently fractionate your protein

B) How to “desalt” your protein following ammonium sulfate precipitation? - Use of gel-filtration in the course mode - Use of dialysis

Major forces within a polypeptide chain and between chains that drive a protein to a stable conformation

-Ion-ion-Ion-dipole (hydrogen bonds)-Hydrophobic

Perturbations of the solvent-protein interactions can cause transitions by disrupting the “old” interactions and promoting formation of new ones.

Perturbations of the solvent-protein interactions

A rise in temperature (can weaken the strength of dipolar interactions and favor formation of hydrophobic interactions)

pH-dependent protonation and deprotonation

Large amount of a salt or organic solvents

Perturbations that cause transitions

Precipitation of proteins by salts

Precipitation occurs by:-neutralization of surface charges by the salt-reducing the chemical activity of the protein-diminishing the effective concentration of water

This is called “salting out” of proteins

Principle behind salting out of proteins

1. The concentration of any salt necessary to cause precipitation of a particular protein is related to the number and distribution of charges and of hydrophobic residues exposed and rendered dominant as the charges are neutralized.

2. This property is exploited to separate some proteins from others by precipitation at high salt concentrations.

3. As salt concentration increases, protein solubility decreases. 4. Net effect is dehydration of proteins which promotes self-association and aggregation

Ammonium sulfate precipitation

©Wilbur H. Campbell, 1996

Ammonium Sulfate Fractionation

Why use ammonium sulfate (NH4)2SO4 as opposed to other salts?

• well tolerated by most proteins

• highly soluble (up to ~ 4 M)

• does alter pH significantly

• relatively inexpensive

Equilibrium Dialysis

VI. Middle Stages – chromatographic separation(s)

What chemical properties of your protein can you takeadvantage of? What is the pI of your protein?

A) Ion-Exchange Chromatography -anion exchange -cation exchange

B) Affinity Chromatography -resin contains ligand or substrate covalently attached -use of an affinity tag (e.g., “His-tag”)

Affinity Chromatography

The basic idea is to use a resin that contains a molecular fragment that interacts specifically withthe species of interest (or a relatively small class ofcompounds that have similar chemical properties).

The species of interest binds to the affinity resin while the other compounds present in the mixture pass through the column. The species of interest can then be eluted.

Affinity Chromatography Illustrated

Affinity Chromatography:another view

Affinity Chromatography Continued: Purification of His-tagged proteins

−from “The QIAexpressionistTM”, by Qiagen, 2003

Two different types of nickelaffinity resins:

NTA – nitrilotriacetic acid

IDA – iminodiacetic acid

−from “The QIAexpressionistTM”, by Qiagen, 2003

Binding of the His-tagged protein to the Ni-NTA resin.

−from “The QIAexpressionistTM”, by Qiagen, 2003

VII. Final (Polishing) Stages

Are there still some nagging contaminants present?

A) Rerunning an ion-exchange column with a slightly different matrix but same functional group

B) Gel-filtration chromatography – high resolution mode

C) Ultra-filtration/centrifugal concentrators

Ultrafiltration

Use centrifugation to achieve solvent flux through the membraneApplications: -Desalting and buffer exchange-Concentrating and purifying proteins, antibodies and nucleic acids (alternative to EtOH precipitation)

Centrifugal Concentrators

Centrifugation forces buffer through the membrane, retaining molecules larger than the cut-off within thesample tube. Repeated dilution and concentration canachieve rapid buffer exchange.

Fundamentals of Ligand Binding:

Ligands collide with their targets, at a rate of kon. Usually this rate is diffusion limited and occurs about a rate of 108 – 109 M−1 s−1.

The ligand leaves its binding site at a rate that depends on the strength of interaction between the ligand and the binding site. Off-rates (koff) range from 106 s−1 (weak binding) to 10−2 s−1 (strong binding). The equilibrium constant for binding is given by:

or

Define the fractional saturation (Y) as follows:

But, since

Y

The binding constant of a protein for a small ligand may be determined by equilibrium dialysis