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How do we purify proteins? BioTic August 5, 2011 Hamline University 1 Outline Protein over-production Preparation of homogenate Purification techniques Ion Exchange Chromatography Affinity Chromatography 2 Think/Pair/Share What would you need to consider before starting a protein purification? Name at least three important points to consider 3 Before starting a purification…. Have a good source of the protein of interest Knowledge about protein composition and properties Why? It will help you decide what purification technique will work best Activity assay to follow the purification of the protein 4 Steps for Protein Purification Isolation of the gene of interest Transfer the gene of interest to suitable organism Transformation Protein production cloning Induction of promoter that controls the gene of interest Cell Lysis Purification How do we choose a purification method? What are the common methods of Protein purification? 5 Getting Enough Protein For protein purification a “rich source” of the protein of interest is needed Can you name some common sources: Tissues Recombinant Cell Extracts 6 Recombinant Cell Extracts Genes encoding the protein of interest are often cloned in bacteria (E.coli) or yeast to facilitate over-production Microbial cells are easy to grow and manipulate Cells lysis is simple making protein extraction fast and convenient 7 Production of Recombinant Proteins 8 How are we over-producing GFP? Beta Lactamase Green Fluorescent Protein (GFP) Ampicillin resistance Aequorea victoria jellyfish gene araC regulator protein Regulates GFP transcription 9 We have cells over producing protein…. What is next? 10 Getting the Protein out the Cells: Preparation of Homogenates Break cells, tissue, or organ Blender, homogenizer, sonication, Microbial cells pressure, or tissue psmotic Supernatant with Soluble protein Pellet with intact cells, organelles, membranes and membrane proteins 11 Separating the Protein of Interest Most common technique Ammonium sulfate precipitation Also known as “salting proteins out” Principle Ammonium sulfate is very polar Binds water molecules easily Competes with proteins for water molecules Eventually proteins lose and “salt out” (precipitate) 12 Ammonium Sulfate Precipitation • Fully hydrated • Solubility depends on Interactions with water molecules • Ammonium sulfate molecules interacting with water molecules ( taking over) • Proteins fall out of solution 13 Further Purification Chromatography Chromatographic separation of proteins are accomplished by several techniques, the most common are: Ion Exchange Chromatography ( IEX) Affinity Chromatography 14 Ion Exchange Chromatography Principle Column filled with a charged resin Negatively charged column ( cation exchanger) Positively charged column ( anion exchanger) Amino acids will bind to the resin according to their ionization state The ionization state of the amino acids will be determined by the pH of the mixture and the pH of the elution buffer 15 Ion Exchange Chromatography 16 Affinity Chromatography Principle Interactions between the protein of interest and a ligand bound to a gel matrix Compounds used for affinity chromatography are usually recognized by the protein of interest Lactate , pyruvate and NAD+ are recognized by lactate dehydrogenase Nickel and histidine tag proteins 17 How are proteins eluted ? Most affinity and ionic exchange columns are eluted using high salt Why? The salt ions compete with the protein for the charges in the column High salt conditions destroy electrostatic interactions holding the protein to the column Affinity columns can be eluted with a substrate or compound that the protein recognizes and can compete with the affinity ligand 18 Laboratory Exercise #5 GFP Purification 19 GFP-Properties Isolated from from Jelly FishAquoera Victoria Function To absorb blue light from another jelly fish protein aequorin Upon absorption of blue light, the GFP emits green light Absorption at 385 nm and emission is at 485 nm QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. 20 QuickTime™ and a TIFF (Uncomp resse d) de com press or are nee ded to s ee this picture. GFP Story QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncomp resse d) d eco mpres sor are nee ded to s ee this picture . Nobel prize in chemistry, 2008 Osamu Shimomura, Martin Chalfie Roger Y.Tsien QuickTime™ and a TIFF (Uncomp resse d) de com press or are nee ded to s ee this picture. QuickTime™ and a and a QuickTime™ (Uncompressed) decompressor TIF FTIFF (Uncompressed) are needed todecompressor see this picture. are needed to see this picture. 21 GFP Structure 11 beta sheets Cylindrical shape The fluorophore is in the center of the structure Known as the “paint can” 22 GFP Fluorophore Originates from an internal ser-tyr-gly sequence Cyclization and post-translational modification of of this tri-peptide allows the formation of a ring system which is resonant and produces fluorescent upon excitation 23 GFP Fluorophore 24 GFP Art QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. 25 GFP Purification Method Hydrophobic interaction Chromatography Why? GFP has long stretches of hydrophobic amino acids in its inner core Upon exposure of the protein to binding buffer, a conformational change occurs and the hydrophobic region is exposed Exposure of the hydrophobic region allows binding of GFP to the hydrophobic resin in the column 26 HIC Macro-Prep Column (Biorad) Methylco-polymer support (beads) In the presence of high salt the hydrophobic groups of the protein interact with the methyl groups in the resin allowing binding Washing with “low salt” buffer allows the less hydrophobic proteins to wash out Final wash with “non-salt buffer”, abolishes hydrophobic interactions and allows the hydrophobic protein to be elute off the column 27 Hydrophobic Interactions Hydrophobic bead O -O S O- O 28 Hydrophobic Interaction Chromatography 1. Add bacterial lysate to column matrix in high salt buffer 2. Wash less hydrophobic proteins from column in low salt buffer 3. Elute GFP from column with no salt buffer 29 Hydrophobic interactions Step #1 Add bacterial lysate to column matrix in high salt buffer Hydrophobic proteins interact with column 30 Step #2 Wash less hydrophobic from column with low salt buffer Less hydrophobic E. coli proteins fall from column GFP remains bound to the column 31 Step#3 Elute GFP from column by adding no salt buffer GFP Released from column matrix Flows through the column 32 Food for thought How would you determine if your GFP purification was effective? Propose two experiments 33