Protein Purification Lecture Note

OUTLINE Protein Purification A. Introduction; what is the basis B. Goals; Specific Activity C. Methods 1. Centrifugation a. b. Differential Isopycnic 2. Precipitation a. b. Salting-out; ammonium sulfate dialysis 3. Chromatography a. b. c. Gel filtration Ion exchange Affinity D. Summary Protein Purification 1 Protein Purification Proteins are separated from each other (along with other macromolecules) due to the vast variability they have. The basis of the separation can be put into 4 categories: • Size, shape, density } } • Charge • Solubility } • Binding characteristics Hydrodynamic properties Chemical properties Biological properties Protein Purification Procedures Basis Procedure Covered Hydrodynamics (size, shape, density Gel filtration Chromatography SDS-PAGE Centrifugation Lab Lab Lab Charge Ion exchange Chromatography Isoelectric focusing Native electrophoresis Lab Solubility Salting out Organic extraction Hydrophobic interaction Chromatography Lab Binding Specificity Affinity Chromatography Lab 2 Before you can separate “your favorite protein (YFP)” from all the thousands of others, you need a way to “see” it.…..... An Assay! • enzymes ➟ activity assay • binding proteins ➟ binding assay • other proteins ➟ immunoblot or ELISA How do you monitor the purification? • Specific Activity = Activity of YFP Total protein How do you measure specific activity? From [protein] (mg/mL) From [activity] (U/mL) Purification of a hypothetical protein Yield (%) 100 96 80 60 45 These are the two important criteria of purifications -If protein was “pure” after step #5, what would the Specific Activity be after you performed a step #6? -What is the Yield? 3 Fractionation by Centrifugation center of rotor Basis = size/shape Differential Speed is ∝ to: Homogenization Relative Centrifugal Force (RCF) = r · w2 g r = radius from center of rotor (mm) w = angular velocity (radians/sec)(rpm·60) g = standard acceleration of gravity (9807 mm/s2) Time is ∝ to: w12 · t1 = w22 · t2 Fractionation by Centrifugation Basis = size/shape Differential Homogenization 4 Fractionation by Centrifugation Basis = density Equilibrium between two opposite forces: Isopycnic (same density) Centrifugal Force Buoyancy Force (less dense) Less dense component More dense component (more dense) Protein Purification Procedures Basis Procedure Covered Hydrodynamics (size, shape, density Gel filtration Chromatography SDS-PAGE Centrifugation Lab Lab Lab Charge Ion exchange Chromatography Isoelectric focusing Native electrophoresis Lab Solubility Salting out Organic extraction Hydrophobic interaction Chromatography Lab Binding Specificity Affinity Chromatography Lab 5 Ammonium Sulfate Fractionation: Fractionation by Salting Out Add (NH4)2SO4 Add more (NH4)2SO4 Basis = solubility least soluble protein middle soluble protein most soluble protein fraction (Pellet fraction) Chapter 3 - Enzyme Purification page 72 Purification in Laboratory begins with Fractionation by Salting Out FLOW CHART FOR LDH PURIFICATION! Bovine heart or muscle tissue! mince, blend, & centrifuge 15 min @ 15,000 rpm ! 1P - precipitate (cell debris, nuclei)! ! !(Crude Extract)! 1S - supernatant fraction* ! ! add (NH4)2SO4 (aq) to 40% saturation! centrifuge 10 min @ 12,000 rpm! ! ! ! 2P - precipitate* ! 2S - supernatant fraction* ! (LDH + other proteins)! (extraneous proteins) ! add (NH4)2SO4 (solid) to 75% saturation! centrifuge 10 min @ 12,000 rpm! ! ! 3P - precipitate* Re-dissolve and dialyze! ! (LDH & other proteins)! ! 3P - dialyzed* ! 3S - supernatant fraction* (extraneous proteins) ! ! ! Assay and load ≤ 5000 units on affinity chromatography column, wash with buffer, then elute with NADH! wash fractions* NADH eluate* (non-specifically bound protein)! ! (LDH-containing fractions)! Pool and concentrate by ultrafiltration! ! Purified LDH*! __________________ *Each fraction marked with an asterisk should be assayed for protein concentration and LDH activity ! 6 Proteins are Least Soluble at Their Isoeletric Point Acidic proteins Notice that between 7.1 – 9.4 most of these proteins are charged Basic proteins If you are performing a salting-out procedure prior to most other procedures, you will need to remove the Molar concentrations of salt. How? Dialysis: Diffusion of Solutes MWCO=molecular weight cutoff Make sure they don’t leak! 7 Protein Purification Procedures Basis Procedure Covered Hydrodynamics (size, shape, density Gel filtration Chromatography SDS-PAGE Centrifugation Lab Lab Lab Charge Ion exchange Chromatography Isoelectric focusing Native electrophoresis Lab Solubility Salting out Organic extraction Hydrophobic interaction Chromatography Lab Binding Specificity Affinity Chromatography Lab Basic Chromatography Liquid phase, mobile phase, eluant } Chromatography “media” ….the column material, beads, gel, etc. = stationary phase, solid phase time time time time eluate Three Types of Chromatography: 1) Gel Filtration (size/shape) 2) Ion exchange (charge) 3) Affinity (function) 8 Gel Filtration Chromatography Basis = size/shape Magnified single bead from the stationary phase of a gel filtration media time time time Smaller molecules latter Larger molecules first Sometimes called a “reverse” sieve Shapes of Proteins: all same scale Recall our picture of all the different sizes and shapes of proteins! 9 Protein Purification Procedures Basis Procedure Covered Hydrodynamics (size, shape, density Gel filtration Chromatography SDS-PAGE Centrifugation Lab Lab Lab Charge Ion exchange Chromatography Isoelectric focusing Native electrophoresis Lab Solubility Salting out Organic extraction Hydrophobic interaction Chromatography Lab Binding Specificity Affinity Chromatography Lab Ion Exchange Chromatography At certain pH values, molecules will have a net charge: • Positive (+) when pH < pI • Negative (–) when pH > pI negatively Negatively charged protein binds to positively charged bead Basis = charge ++ + ++ + + + + + + + + + Positively charged protein flows through Negatively charged particles/beads = cat-ion exchange Positively charged particles/ beads = anion exchange 10 Ion Exchange Chromatography Will bind to Cation exchanger If pH < pI POSITIVE charge ubiquitin Will bind to Anion exchanger pH < pI > pI Net charge of protein + – Sticks to – (cation exchanger) + (anion exchanger) Types Dowex-50, carboxylmethyl (CM), Phosphocellulose, mono-S (methyl-sulfite) Dowex-1, Diethylaminoethyl (DEAE), mono-Q (quaternary amine) If pH > pI NEGATIVE charge Ion Exchange Chromatography Basis = charge What can you use to elute proteins that stick? time Switch eluant time Elute with salt or pH gradient 11 Ion Exchange Chromatography: EXAMPLE of an amino-acid mixture Basis = charge cation If this were a mixture of G, K, and D, what would the order of elution be? 1st= D, 2nd = G, 3rd = K Protein Purification Procedures Basis Procedure Covered Hydrodynamics (size, shape, density Gel filtration Chromatography SDS-PAGE Centrifugation Lab Lab Lab Charge Ion exchange Chromatography Isoelectric focusing Native electrophoresis Lab Solubility Salting out Organic extraction Hydrophobic interaction Chromatography Lab Binding Specificity Affinity Chromatography Lab 12 Affinity Chromatography Basis = function G G Affinity Chromatography Biotechnology (recombinant DNA technology) has revolutionized protein purification. Basis = function At the level of the DNA sequence, the DNA sequence encoding such binding proteins or ”tags” can be “fused” to the sequence encoding YFP. In this way, a chimeric protein is produced that has the binding function, which allows the use of affinity chromatography. Common “tags” are: Column beads have attached: Maltose-binding protein Maltose Chitin-binding protein Chitin Glutathione-S-transferase Glutathione (g-Glu-Cys-Gly) His-His-His-His-His-His Ni-chelate 13 Protein Purification Procedures Basis Procedure Covered Hydrodynamics (size, shape, density Gel filtration Chromatography SDS-PAGE Centrifugation Lab Lab Lab Charge Ion exchange Chromatography Isoelectric focusing Native electrophoresis Lab Solubility Salting out Organic extraction Hydrophobic interaction Chromatography Lab Binding Specificity Affinity Chromatography Lab Purification of Myoglobin (Mb) Overall 0.00048 0.00064 47 0.056 37 1.3 117 Calculations: Specific Activity Yield Fold Purification 0.75 ÷ 1550 = 0.00048 0.35 ÷ 550 = 0.00064 0.35 ÷ 0.75 = 0.47 0.00064 ÷ 0.00048 = 1.3 0.28 ÷ 5.0 = 0.056 0.28 ÷ 0.75 = 0.37 0.056 ÷ 0.00048 = 117 14 Purification of a hypothetical protein Fold increase Yield in SA (%) } 3x } } } 100 96 6x 3x 80 60 25x 45 % loss in Yield } } } } 4% 17% 25% 25% Calculate fold increase in SA for each step ➔ helps determine if step is effective. ......step 5 Which is the best step? ......step 2 or step 4 Which is the worst step? Look at yield…... Calculate fraction (%) of YFP lost at each step. ......step 4 Which of step 2 or step 4 resulted in loss of more YFP? Purification of a hypothetical protein Fold increase Yield in SA (%) 100 96 1500x 80 60 45 Always increasing until pure Overall purification = 1500x Estimate Approximate Expression Level: Yield of 45% means you can calculate the mg if 100%: It would be 3/0.45 = 6.7 mg of YFP If total protein was 10,000 mg, then fraction that is YFP in crude is: 6.7/10000=0.00067=0.07% Well….... 1/(overall purification) gives the same result (1/1500=0.07%) 15