Protein Purification 9/21/16. Protein Purification Procedures

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1 Protein Purification 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 Protein Purification Procedures Centrifugation 1

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 Specific Activity = Activity of YFP Total protein What is the Yield? blue blue blue From [protein] (mg/ml) From [activity] (U/mL) Purification of a hypothetical protein Yield (%) What is the Yield? What would the Specific Activity be if protein was pure and you performed a step #6? 2

3 Purification of a hypothetical protein Yield (%) What is the Yield? What would the Specific Activity be if protein was pure and you performed a step #6? Fractionation by Centrifugation Differential center of rotor size/shape Homogenization Speed is to: Relative Centrifugal Force (RCF) = r w 2 g r = radius from center of rotor (mm) w = angular velocity (radians/sec)(rpm 60) g = standard acceleration of gravity (9807 mm/s 2 ) Time is to: w 12 t1 = w 22 t2 Fractionation by Centrifugation Differential size/shape Homogenization 3

4 Fractionation by Centrifugation Isopycnic (same density) Equilibrium between two opposite forces: Centrifugal Force Buoyancy Force density (less dense) (more dense) (less dense) (more dense) Fractionation by Centrifugation Isopycnic (same density) Equilibrium between two opposite forces: Centrifugal Force Buoyancy Force density (less dense) (more dense) (less dense) (more dense) Ammonium Sulfate Fractionation: Fractionation by Salting Out solubility Add (NH4)2SO4 Add more (NH4)2SO4 least soluble protein middle soluble protein most soluble protein fraction (Pellet fraction) 4

5 Purification in Laboratory begins with Fractionation by Salting Out FLOW CHART FOR LDH PURIFICATION! Bovine heart or muscle tissue! mince, blend, & centrifuge 15 15,000 rpm! 1P!- precipitate! 1S!- supernatant fraction*! (cell debris, nuclei)! (Crude Extract)! add (NH 4) 2SO 4 (aq) to 40% saturation! centrifuge 10 12,000 rpm!! 2P! - precipitate*! 2S! - supernatant fraction*! (extraneous proteins)! (LDH + other proteins)! add (NH 4) 2SO 4 (solid) to 75% saturation! centrifuge 10 12,000 rpm!! 3P! - precipitate*! 3S! - supernatant fraction*! (LDH & other proteins)! (extraneous proteins)! Re-dissolve and dialyze! 3P!- dialyzed*! 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)!! Purified LDH*! Pool and concentrate by ultrafiltration! Proteins are Least Soluble at Their Isoeletric Point Acidic proteins Notice that between 7 9 most proteins are charged Basic proteins Dialysis: Diffusion of Solutes Make sure they don t leak! 5

6 Basic Chromatography Chromatography media.the column material, beads, gel, etc. = stationary phase, solid phase Liquid phase, mobile phase, eluant eluate Three Types of Chromatography: 1) Gel Filtration (size/shape) 2) Ion exchange (charge) 3) Affinity (function) Gel Filtration Chromatography size/shape Larger molecules first Gel Filtration Chromatography size/shape Larger molecules first 6

7 Shapes of Proteins: all same scale 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 charge Positively charged protein flows through Negatively charged particles, beads = cation exchange Positively charged particles, beads = anion exchange Aldolase ph < pi > pi Net charge + Sticks to (cation exchanger) + (anion exchanger) Types Dowex-50, carboxylmethyl (CM), Phosphocellulose, mono-s Dowex-1, Diethylaminoethyl (DEAE), mono-q (quaternary amine) 7

8 Ion Exchange Chromatography charge Elute with salt or ph gradient Ion Exchange Chromatography: Example of amino-acid mixture cation charge If this were a mixture of G, K, and D, what would the order of elution be? 1 st = D, 2 nd = G, 3 rd = K Affinity Chromatography function G G 8

9 Affinity Chromatography function G G Affinity Chromatography Biotechnology (recombinant DNA technology) has function revolutionized protein purification. 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: Maltose-binding protein Chitin-binding protein Glutathione-S-transferase His-His-His-His-His-His Column beads have attached: Maltose Chitin Glutathione (g-glu-cys-gly) Ni-chelate Purification of Myoglobin (Mb) Calculations: Specific Activity = = = Yield = = 0.37 Fold Purification = = 117 9

10 Purification of a hypothetical protein Fold increase in SA Yield (%) % loss in Yield 3x 6x 3x 25x % 17% 25% 25% Calculate fold increase in SA for each step helps determine if step is effective. Which is the best step?...step 5 Which is the worst step?...step 2 or step 4 Look at yield... Calculate fraction (%) of YFP lost at each step. Which of step 2 or step 4 resulted in loss of more YFP?...step 4 Purification of a hypothetical protein Fold increase in SA Yield (%) x Always increasing 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= =0.07% Well... 1/(overall purification) gives the same result (1/1500=0.07%) 10