Protein purification chp-5-bioc-361-version-oct-2012
Mary K. Campbell Shawn O. Farrell http://academic.cengage.com/chemistry/campbell Chapter Five Protein Purification andCharacterization Techniques Paul D. Adams • University of Arkansas
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
How pure should my protein be? Application Required PurityTherapeutic use, in vivo Extremely high > 99%studiesBiochemical assays, X-ray High 95-99%crystallographyN-terminal sequencing,antigen for antibody Moderately high < 95%production, NMR
Separation of proteins based on physicaland 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 ActivityEnzyme 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-1Specific 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.
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
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.
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)
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 pointSilver 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