Protein Purification Biochem 4511 Figures Essentials of Biochemistry 3rd Ed OSU Custom Edition Principles of Biochemistry 5th Ed Moran et al Lehninger Principles of Biochemistry 5th Ed Nelson Cox Fundamentals of Biochemistry 2nd Ed Voet Voet Pratt Protein Purification 1 Overexpress the protein Bacterial cells yeast mammalian cell culture etc 2 Break open lyse the cells to extract the protein 3 Perform selective separation steps until the protein is isolated to sufficient purity Protein Separation Most physical properties of proteins may be utilized to separate and purify proteins Choose properties that characterize your protein of interest and separate from other proteins based on these properties Solubility pellet or supernatant Ionic charge ion exchange electrophoresis Polarity Hydrophobicity hydrophobic interaction chromatography Size gel filtration chromatography dialysis Binding specificity affinity purification Gel Electrophoresis Molecules with a net charge will migrate in an electric field with a velocity proportional to 1 Charge density 2 Size 3 Shape Zonal electrophoresis Constrain ions to move within a solid support such as a thin gel between glass plates to minimize diffusion Gel electrophoresis is generally used as an analytical technique a preparative technique PAGE Polyacrylamide Gel Electrophoresis PAGE Polyacrylamide Gel Electrophoresis Extra Polymerized and cross linked acrylamide mesh controlled by amount of acrylamide and degree of crosslinking PAGE Polyacrylamide Gel Electrophoresis Large proteins tangle in the gel and have slower migration Density of the cross linked gel determines the size range for proteins that move well through gel SDS PAGE Denaturing Gel Electrophoresis SDS PAGE SDS PolyAcrylamide Gel Electrophoresis SDS sodium dodecyl sulfate detergent with C12 hydrocarbon chain and negatively charged sulfate SDS denatures protein tertiary structure by disrupting internal charge and hydrophobic interactions uniform shape Overwhelms protein native charge uniform negative charge Result Mobility of proteins towards the positive electrode depends only on size SDS PAGE Sample Preparation Preparing sample for SDS PAGE denaturing gel electrophoresis SDS sodium dodecyl sulfate denaturing detergent coats protein with negative charge Reducing agent mercaptoethanol dithiothreitol break any disulfide bonds Blue dye to visualize the solvent front Glycerol increases density of sample so it drops to the bottom of the sample well Boil for 10 minutes to ensure maximal unfolding SDS PAGE Sample Preparation Denaturation Denaturation Add SDS and heat to disrupt tertiary structure and impart a uniform negative charge All proteins now have a uniform charge mass ratio SDS Heat Folded Unfolded SDS PAGE Sample Preparation Reduction Reduction of covalent protein disulfide bonds Common reducing agents BME TCEP 1 4 dithiothreitol mercaptoethanol tris 2 carboxyethyl phosphine SDS PAGE 1 SDS unfolds the protein uniform shape 2 SDS coats all of the proteins with negative charge uniform charge density Only the size remains to differentiate the proteins SDS PAGE Determining Molecular Weight Measure and plot distance migrated of protein standards vs log of their molecular weights MW Draw a standard curve through the linear range Measure the distance migrated of your Unknown Locate the corresponding log MW and find the MW of the Unknown protein SDS PAGE Protein visualization Molecular Weight Ladders Protein visualization Coomassie Brilliant Blue dye complexes with the unfolded proteins detects 100 500 ng Western blotting Detect a specific protein by staining with an antibody Sensitivity depends on antibody but can be 1 ng Coomassie Blue Stained Gel PAGE Polyacrylamide Gel Electrophoresis Denaturing gels SDS PAGE typical example Denature proteins break up complexes break down disulfide bonds then run gel Use to show what proteins are present in a mixture Native gels Run electrophoresis under conditions that maintain folded proteins complexes associations Can be used to demonstrate that proteins bind to each other or to DNA SDS PAGE vs PAGE Native PAGE of DNA and histone octamer complex DNA Only Protein DNA Nuc DNA SDS PAGE of 7 different folded histone octamers H3 H2A H2B H4 Figures Ottesen Lab Column Chromatography Column Chromatography uses interactions between a protein mixture and a solid support for separation based on a single physical property Different physical properties can be used Elution The process of flowing the protein out of the column Types of Chromatography Stationary phase column Ion exchange Charged stationary phase Gel filtration size based Reverse phase Hydrophobic stationary phase Affinity specific interactions Mobile phase solvent conditions required to elute protein depends on protein and column used Size of column purification or sample analysis For ion exchange Isoelectric Point pI In solution folded proteins look like big clusters of ionic charges Each protein has an isoelectric point pI which is the pH at which the protein carries no net charge Technically pI pK1 pK2 for transitions around neutral When pH pI protein will be negatively charged When pH pI protein will be positively charged The minimal solubility of a folded protein comes where pH pI for that protein because of interactions with water as solvent Isoelectric Point pI Environment influences the pKa of ionizable groups by stabilizing or destabilizing the charged state Calculated protein pI is not usually accurate beyond 1 pH unit environment affects side chain pKa To be accurate the value must be measured But estimates can be useful Ion Exchange Chromatography Separation Principle Electrostatic interactions between a charged protein above or below the pI and a charged stationary phase A positively charged protein basic will bind to a negatively charged matrix acidic A negatively charged protein will bind to a positively charged matrix Increasing the concentration of ions in solution increasing salt concentration will elute the protein from the column Ion Exchange Chromatography Common terminology for ion exchange resins Anion exchange positively charged basic matrix that interacts with anions that interacts with cations Cation exchange negatively charged acidic matrix Diethylaminoethyl Carboxymethyl Ion Exchange Chromatography Choose pH where protein has appropriate charge Protein will interact with bead through electrostatics Salt Na or Cl will compete for interactions and exchange the ions A