Paul D. Adams • University of Arkansas

Mary K. CampbellShawn O. Farrellhttp://academic.cengage.com/chemistry/campbell

Chapter FiveProtein Purification and

Characterization Techniques

Why purify a protein?

• Characterize function, activity, structure

• Use in assays

• Raise antibodies

• many other reasons ...

Guidelines for protein purification

• Define objectives• Define properties of target protein and critical

contaminants• Minimize the number of steps• Use a different technique at each step• Develop analytical assays

Adapted from: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC

How pure should my protein be?

Application Required Purity

Therapeutic use, in vivo studies

Extremely high > 99%

Biochemical assays, X-ray crystallography

High 95-99%

N-terminal sequencing, antigen for antibody production, NMR

Moderately high < 95%

Separation of proteins based on physical and chemical properties

• Solubility

• Binding interactions

• Surface-exposed hydrophobic residues

• Charged surface residues

• Isoelectric Point

• Size and shape

The overall goal

• To remove as much of the “other” protein as possible and keep as much of your target protein as possible

• This is a great challenge since at each step you sacrifice some of your target protein.

• Activity = total target protein activity in your sample• Specific activity = how much target enzyme activity

you have with respect to total protein content present

• Which number should go up and which down?

Activity versus Specific Activity

Enzyme activity• Enzyme activity = moles of substrate converted per

unit time = rate × reaction volume. Enzyme activity is a measure of the quantity of active enzyme present

• 1 enzyme unit (U) = 1 μmol min-1

Specific activity• The specific activity is the activity of an enzyme per

milligram of total protein • expressed in μmol min-1mg-1. • Specific activity is equal to the rate of reaction x

volume of reaction / mass of total protein.

How We Get Proteins Out of Cells

Proteins/enzymes are delicate

• Remember Proteins are delicate and subject to denaturation.• Often tracking a protein based on its activity or function

therefore it needs proper conformation• Cells are full of hydrolytic enzymes when you fracture or lyse a

cell proteins and enzymes are mixed and degradation occurs immediately

• Keep things cold (on ice)• Add protease inhibitors

• Many considerations to be made when using and selecting protease inhibitors – remember the six classes of enzymes – don’t want to inhibit and enzyme activity when need to assay during the purification

How will you track your protein?

• Purification is often a multi-step process• You need to track or “assay for your protein” after each

step

• If it is an enzyme you can test for its activity

• If you have an antibody you can use Western blot or ELISA

• You can test for its size (not as specific)

• You could use mass spectrometry to identify it

• You could use N-terminal sequencing to ID the traget protein

Salting Out

• After Proteins solubilized, they can be purified based on solubility (usually dependent on overall charge, ionic strength, polarity

• Ammonium sulfate (NH4SO4) commonly used to “salt out”

• Takes away water by interacting with it, makes protein less soluble because hydrophobic interactions among proteins increases

• Different aliquots taken as function of salt concentration to get closer to desired protein sample of interest (30, 40, 50, 75% increments)

• One fraction has protein of interest

Column Chromatography

• Basis of Chromatography

• Different compounds distribute themselves to a varying extent between different phases

• Interact/distribute themselves

• In different phases

• 2 phases:

• Stationary: samples interacts with this phase

• Mobile: Flows over the stationary phase and carries along with it the sample to be separated

Column Chromatography

Ion Exchange

• Interaction based on overall charge (less specific than affinity)

• Cation exchange

• Anion exchange

Size-Exclusion/Gel-Filtration

• Separates molecules based on size.

• Stationary phase composed of cross-linked gel particles.

• Extent of cross-linking can be controlled to determine pore size

• Smaller molecules enter the pores and are delayed in elution time. Larger molecules do not enter and elute from column before smaller ones.

Size Exclusion/Gel-filtration (Cont’d)

Affinity Chromatography

•Uses specific binding properties of molecules/proteins

•Stationary phase has a polymer that can be covalently linked to a compound called a ligand that specifically binds to protein

Electrophoresis

• Electrophoresis- charged particles migrate in electric field toward opposite charge

• Proteins have different mobility:

• Charge

• Size

• Shape

• Agarose used as matrix for nucleic acids

• Polyacrylamide used mostly for proteins

Electrophoresis (Cont’d)

• Polyacrylamide has more resistance towards larger molecules than smaller

• Protein is treated with detergent (SDS) sodium dodecyl sulfate

• Smaller proteins move through faster (charge and shape usually similar)

SDS PAGE – to track your purification

Isoelectric Focusing

• Isolectric focusing- based on differing isoelectric pts. (pI) of proteins

• Gel is prepared with pH gradient that parallels electric-field. What does this do?

• Charge on the protein changes as it migrates.

• When it gets to pI, has no charge and stops

2D gel – Size and Isoelectric point

Silver or commassie blue stain ---- Sypro Ruby - fluorescent

Differential Centrifugation

• Sample is spun, after lysis, to separate unbroken cells, nuclei, other organelles and particles not soluble in buffer used

• Different speeds of spin allow for particle separation