Chapter 5 - Protein Purification - Tagged 2.pdf

CHAPTER 5: PROTEIN PURIFICATION & CHARACTERIZATION Week5 CHEM311 Summary Extraction Purification Column chromatography; gel, affinity and ionexchange Electrophoresis: SDS-page and isoelectric focussing Characterization (primary structure determination) Digestion & the Edman Method Mass spectrometry...

CHAPTER 5: PROTEIN PURIFICATION & CHARACTERIZATION Week5 CHEM311 Summary Extraction Purification Column chromatography; gel, affinity and ionexchange Electrophoresis: SDS-page and isoelectric focussing Characterization (primary structure determination) Digestion & the Edman Method Mass spectrometry Proteomics Why purify a protein? Characterize function, activity, structure Use in assays Raise antibodies Medicines (Insulin, EPO, Botulinum toxin (BoTox)… 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 Therapeutic use, in vivo studies Required Purity 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 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 Extraction of Proteins from Cells Many different proteins exists within one cell Many steps needed to extract protein of interest, and separate from many contaminants Before purification begins, protein must be released from cell by homogenization (physical disruption of cell to release proteins; lysis of cells) How We Get Proteins Out of Cells How We Get Proteins Out of Cells Homogenization (lysis) techniques Differential centrifugation to recover clean protein fraction 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 Salting Out After Proteins are solubilized, they can be purified based on solubility (usually dependent on overall charge, ionic strength, polarity Ammonium sulfate (NH4)2SO4 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 Purification using column chromatography Basis of Chromatography Different compounds distribute themselves to a varying extent between different phases Proteins in solution interact/distribute themselves into two different phases: Stationary (resin in column) Mobile (solvent passing through column) 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 Size-Exclusion/Gel-Filtration Chromatography 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 Size Exclusion/Gel-filtration (Cont’d); Use of a gel that doesn’t interact with protein – agarose, polyacrylamide, starch or dextran Column Chromatography Ion Exchange https://www.youtube.com/watch?v =Z54ec_G12QE Ion Exchange Chromatography Interaction based on overall charge (less specific than affinity) Cation exchange: Bind to +vely charged groups Anion exchange Bind to –vely charged groups Cationic exchange using a gradient of increasing concentration of NaCl as eluent Ion Exchange Chromatography (1)At the beginning of the separa3on, various proteins are applied to the column. The column resin is bound to Na+ counterions (small red spheres). (2) Proteins that have no net charge or a net nega3ve charge pass through the column. Proteins that have a net posi3ve charge s3ck to the column, displacing the Na+. (3) An excess of Na+ ion is then added to the column. (4) The Na+ ions outcompete the bound proteins for the binding sites on the resin, and the proteins elute. Affinity Chromatography Uses specific binding properties of molecules/proteins Stationary phase has a resin that can be covalently linked to a compound called a ligand that specifically binds to protein Group Specific Affinity Resins