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Lecture 8: Protein purification –Protein purification.

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1 Lecture 8: Protein purification –Protein purification

2 Column chromatography After the initial fractionation steps we move to column chromatography. The mixture of substances (proteins) to be fractionated is dissolved in a liquid or gaseous fluid called the mobile phase. This solution is passed through a column consisting of a porous solid matrix called the stationary phase. These are sometimes called resins when used in liquid chromatography. The stationary phase has certain physical and chemical characteristics that allow it to interact in various ways with different proteins. Common types of chromatographic stationary phases –Ion exchange –Hydrophobic –Gel filtration –Affinity

3 Ion exchange chromatography Ion exchange resins contain charged groups. If these groups are acidic in nature they interact with positively charged proteins and are called cation exchangers. If these groups are basic in nature, they interact with negatively charged molecules and are called anion exchangers. DEAE cellulose anion exchanger CH 2 -CH 2 -NH + (CH 2 CH 2 ) Negatively charged (acidic) protein or enzyme CM cellulose cation exchanger CH 2 -COO Positively charged (basic) protein or enzyme CH 2 -CH 2 -NH + (CH 2 CH 2 )

4 Ion exchange chromatography CM cellulose cation exchanger CH 2 -COO Positively charged protein or enzyme bind to the column For protein binding, the pH is fixed (usually near neutral) under low salt conditions. Example cation exchange column… Negatively charged proteins pass through the column

5 Ion exchange chromatography CM cellulose cation exchanger CH 2 -COO To elute our protein of interest, add increasingly higher amount of salt (increase the ionic strength). Na + will interact with the cation resin and Cl - will interact with our positively charged protein to elute off the column. CM cellulose cation exchanger CH 2 -COO Na + Cl - Na +2 Cl - Na +2 + Increasing [NaCl] of the elution buffer

6 Ion exchange chromatography Proteins will bind to an ion exchanger with different affinities. As the column is washed with buffer, those proteins relatively low affinities for the ion exchange resin will move through the column faster than the proteins that bind to the column. The greater the binding affinity of a protein for the ion exchange column, the more it will be slowed in eluting off the column. Proteins can be eluted by changing the elution buffer to one with a higher salt concentration and/or a different pH (stepwise elution or gradient elution). Cation exchangers bind to proteins with positive charges. Anion exchangers bind to proteins with negative charges.

7 Figure 6-6Ion exchange chromatography using stepwise elution. Page 134

8 Ion exchange chromatography Gradient elution can improve the washing of ion exchange columns. The salt concentration and/or pH is continuously varied as the column is eluted so as to release sequentially the proteins bound to the column. The most widely used gradient is the linear gradient where the concentration of eluant solution varies linearly with the volume of the solution passed. The solute concentration, c, is expressed as c = c 2 - (c 2 - c 1 )f c 1 = the initial concentration of the solution in the mixing chamber c 2 = the concentration of the reservoir chamber f = the remaining fraction of the combined volumes of the solutions initially present in both reservoirs.

9 Figure 6-7Device for generating a linear concentration gradient. Page 135 c = c 2 - (c 2 - c 1 )f

10 Figure 6-8Molecular formulas of cellulose- based ion exchangers. Page 135

11 Table 6-2Some Biochemically Useful Ion Exchangers.

12 Ion exchange chromatography Ion exchangers can be cellulosic ion exchangers and gel-type ion exchangers. Cellulosic ion exchangers most common. Gel-type ion exchangers can combine with gel filtration properties and have higher capacity. Disadvantage-these materials are easily compressed so eluant flow is low. There are other materials derived from silica or coated glass beads that address this problem.

13 Gel filtration chromatography Also called size exclusion chromatography or molecular sieve chromatography. How does it work? If we assume proteins are spherical… size Molecular mass (daltons) 10,000 30, ,000

14 Gel filtration chromatography flow

15 Gel filtration chromatography flow

16 Gel filtration chromatography flow

17 Gel filtration chromatography flow

18 Gel filtration chromatography flow

19 Gel filtration chromatography The molecular mass of the smallest molecule unable to penetrate the pores of the gel is at the exclusion limit. The exclusion limit is a function of molecular shape, since elongated molecules are less likely to penetrate a gel pore than other shapes. Behavior of the molecule on the gel can be quantitatively characterized. Total bed volume of the column V t = V x + V 0 V x = volume occupied by gel beads V 0 = volume of solvent space surrounding gel; Typically 35%

20 Gel filtration chromatography Elution volume (V e ) is the volume of a solvent required to elute a given solute from the column after it has first contacted the gel. Relative elution volume (V e /V 0 ) is the behavior of a particular solute on a given gel that is independent of the size of the column. This effectually means that molecules with molecular masses ranging below the exclusion limit of a gel will elute from a gel in the order of their molecular masses with the largest eluting first.

21 Figure 6-9Gel filtration chromatography. Page 137

22 Figure 6-10Molecular mass determination by gel filtration chromatography. Page 138

23 Table 6-3Some Commonly Used Gel Filtration Materials. Page 138

24 Gel filtration chromatography Elution volume (V e ) is the volume of a solvent required to elute a given solute from the column after it has first contacted the gel. Relative elution volume (V e /V 0 ) is the behavior of a particular solute on a given gel that is independent of the size of the column. This effectually means that molecules with molecular masses ranging below the exclusion limit of a gel will elute from a gel in the order of their molecular masses with the largest eluting first.

25 Affinity chromatography Many proteins can bind specific molecules very tightly but noncovalently. We can use this to our advantage with affinity chromatography. Glucose (small dark blue molecule) binding to hexokinase. The enzyme acts like a jaw and clamps down on the substrate (glucose)

26 Affinity chromatography How does it work? Ligand - a molecule that specifically binds to the protein of interest. Inert support Spacer arms + + Ligand Inert support Affinity material prepared

27 Affinity chromatography Inert support Mixture of proteins Inert support Unwanted proteins

28 Affinity chromatography Inert support Remove from competitive ligand by dialysis. Inert support Elute with competitive ligand.

29 Affinity chromatography To remove the protein of interest from the column, you can elute with a solution of a compound with higher affinity than the ligand (competitive) You can change the pH, ionic strength and/or temperature so that the protein-ligand complex is no longer stable.

30 Immunoaffinity chromatography Monoclonal antibodies can be attached to the column material. The column only binds the protein against which the antibody has been raised. 10,000-fold purification in a single step! Disadvantges –Difficult to produce monoclonal antibodies (expensive $$!) –Harsh conditions to elute the bound protein

31 Other chromatographic methods Adsorption chromatography - nonpolar molecules physically adosrbed on the surface of an insoluble substance (alumina, diatomeceous earth, silica gel, etc.) through Van der Waals forces. Molecules eluted from the column by organic solvents (chloroform, hexane, ethyl ether). Based on the partition of polar column material and nonpolar solvent. Not used often with proteins. Hydroxyapatite chromatography - gels of crystalline hydroxyapatite (an insoluble form of calcium phosphate) adsorb proteins. Separation occurs with a gradient elution of the column with phosphate buffer.

32 Other chromatographic methods Paper chromatography - separation of small polar molecules. Mostly used to separate amino acids, oligopeptides. Historically the first chromatography but not really used today. However, principles of its use are useful to know. Rates of migration of the substances are determined by relative solubilities in the polar stationary phase (paper) and the nonpolar mobile phase A given solute is distributed between the mobile and stationary phases according to its partition coefficient K p = concentration in stationary phase concentration in mobile phase Molecules are separated according to their polarities, with nonpolar molecules moving faster than polar molecules

33 Other chromatographic methods After the solvent has migrated an appropriate distance, the chromatogram is removed from the solvent and dried. If not colored, the separated materials can be detected by radioactivity, fluroescence, etc. The migration rate of the substance is expressed by the following ratio: Each substance has a characteristic R f value for a given solvent and paper type. Reverse-phase chromatography (RPC)- separates nonpolar substances including denatured proteins. Form of liquid-liquid partition chromatography in which the polar character of the phases is reversed relative to paper chromatography. Stationary phase is nonpolar and the mobile phase is a more polar liquid. Used to separate lipids but can also be used for proteins. Solvent must be highly nonpolar usually high concentration of organic solvent (acetonitrile) so it denatures proteins so that the hydrophobic cores can interact with the matrix. R f = Distance traveled by substance Distance traveled by solvent front

34 Other chromatographic methods Hydrophobic interaction chromatography (HIC)- the stationary phase is hydrophillic (agarose gel) with substituted hydrophobic groups. Interactions with column are relatively weak and can be used for the separation of native proteins (not denatured), so proteins are separated based on surface hydrophobicity. High performance liquid chromatography (HPLC)- may be based on adsorption, ion exchange, size exclusion, HIC or RPC but is improved because of the noncompressible matrix. Can be made of silica and withstand very high pressures (up to 5000 psi) so flow rates can be very high. Advantages of HPLC –High resolution –Fast –High sensitivity –Can be easily automated

35 Dialysis Dialysis-a process that separates molecules according to size through the use of semipermeable membranes containing pores of less than macromolecular dimensions. Pores in the membrane allow solvents, salts and small metabolites to diffuse across but block larger molecules. Cellophane (cellulose acetate) most commonly used dialysis material. Usually used to change the solvent in which the protein is dissolved in. Can also be used to concentrate a protein solution by placement in a polymeric dessicant (PEG) which cannot go through the membrane but absorbs water through the membrane.

36 Figure 6-11Use of dialysis to separate small and large molecules. Page 139

37 Table 6-4Purification of Rat Liver Glucokinase. Page 142

38 Electrophoresis The migration of ions in an electric field to separate molecules. Many forms of electrophoresis-we will focus on polyacrylamide gel electrophoresis (PAGE). PAGE techniques are often used determine the purity of proteins.

39 Figure 6-20Apparatus for slab gel electrophoresis. Page 146

40 Figure 6-21Diagram of a disc electrophoresis apparatus. Page 147

41 Sodium dodecyl sulfate (SDS-PAGE) Native protein is unfolded by heating in the presence of  - mercaptoethanol and SDS. SDS binds to the protein so that it stays in solution and denatures. Large polypeptides bind more SDS than small polypeptides, so proteins end up with negative charge in relation to their size. Thus, we can separate the proteins based on their mass. Native protein Heat + Reductant + SDS Denatured protein with bound SDS N C

42 Figure 6-24SDS-PAGE. Page 149

43 Figure 6-25Logarithmic relationship between the molecular mass of a protein and its relative electrophoretic mobility in SDS-PAGE. Page 149

44 Figure 6-23Detection of proteins by immunoblotting. Page 148

45 Isoelectric focusing For looking at proteins without charge, proteins can be treated with 6M urea (denatures but unlike SDS does not put charges on a protein). Thus, a mixture of proteins can be electrophoresed through a solution having a a stable pH gradient in from the anode to the cathode and a each protein will migrate to the position in the pH gradient according to its isoelectric point. This is called isoelectric focusing. Ampholytes (amphoteric electrolytes)-low molecular mass ( D) ooligomers with aliphatic amino and carboxylic acid groups with a range of isoelectric points. Ampholytes help maintain the pH gradiennt in the presence of high voltage. Can also use gels with immobilized pH gradients -made of acrylamide derivatives that are covalently linked to ampholytes. Used with a gradient maker to ensure continuously varied mixture when the gel is made.

46 Figure 6-26General formula of the ampholytes used in isoelectric focusing. Page 150

47 Isoelectric focusing 2D-gel electrophoresis is an invalubale tool for proteomics. Proteome (like genome) is the total number of all proteins expressed by a cell or organism, but with an emphasis on their quantitation, localization, modifications, interactions, and activities, as well as their identification. Individual protein bands froma stained gel can be cut out of a gel, destained, and and the protein can be eluted from the gel fragment for identification and characterization using mass spec.

48 Figure 6-27 Two-dimensional (2D) gel electrophoresis. Page 150

49 Table 6-1Isoelectric Points of Several Common Proteins. Page 133

50 Summary of techniques for protein purification Cell lysis techniques - osmolysis, mechanical disruption-high speed blender, homogenizer, French press, sonication Salting out and salting in Chromatography –Ion exchange –Size exclusion –Affinity –others Dialysis Electrophoresis –SDS PAGE Isoelectric focusing

51 Crystallization Page 133

52 Crystallization Crystallization of proteins- difficult. Protein must be homogeneous (e.g. pure) Supersaturated solution prepared (10 mg/ml) and allowed to stand until crystals form. Use x-ray diffraction to observe the bonds that hold the 3-D shape of the protein.

53 3-D structure of proteins X-ray source Single crystal of protein Diffraction pattern Computational recombination of scattered x-rays Electron density map Structural model

54 Figure 8-35X-Ray diffraction photograph of a single crystal of sperm whale myoglobin.

55 Figure 8-36aElectron density maps of proteins. Page 241

56 Figure 8-36bElectron density maps of proteins. Page 241

57 Figure 8-36cElectron density maps of proteins. Page 241

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