Chapter 6 Techniques of Protein Purification You should become familiar with this entire chapter so that you can use it for reference. But you are only responsible for the topics in the chapter that were covered in lecture.
Protein isolation Selection of protein source • Tissues from animals • Microorganisms (E. coli or yeast) • Molecular cloning techniques Methods of solubilization • Osmosis lysis (with hypotonic solution) • Use of lysozyme (enzyme that degrades cell wall) • French press or sonication
Stabilization of proteins • 1. pH (think buffers!) • 2. Temperature (close to 0oC) • Thermal stability could be used for purification • 3. Addition of protease inhibitors • 4. Gentle handling (no frothing) • Assay of proteins • 1. If purifying an enzyme, use the • reaction it catalyzes as an assay • 2. If metalloprotein use the metals to follow the protein • 3. Immunochemical techniques (antibodies)
General strategy of protein purification Proteins are purified by fractionation procedures, a series of independent steps in which the properties of protein of interest are utilized to separate it from other contaminating proteins. How do we know our sample of protein is pure? We don't! The best we can do is to demonstrate by all available methods that our sample consists of only one component.
General strategy of protein purification CharacteristicProcedure Solubility 1. Salting in 2. Salting out Ionic charge:1. Ion exchange chromatography 2. Electrophoresis 3. Isoelectric focusing Polarity: 1. Adsorption chromatography 2. Paper chromatography 3. Hydrophobic interaction chromatography Molecular size: 1. Dialysis and ultrafiltration 2. Gel electrophoresis 3. Gel filtration chromatography 4. Ultracentrifugation Binding specificity: 1. Affinity chromatography
Solubility of a protein in aqueous solution Depends strongly on: Concentrations of dissolved salts pH Temperature 4. Addition of water-miscible organic solvents, e.g., ethanol or acetone
Solubility of carboxyhemoglobin at its isoelectric point as a function of ionic strength and ion type Page 131
Solubilities of several proteins in ammonium sulfate solutions Page 131
Solubility of b-lactoglobin as a function of pH at several NaCl concentrations Page 132
Isoelectric Points of Several Common Proteins Protein pI Pepsin 1.0 Ovalbumin (hen) 4.6 Serum albumin (human) 4.9 Tropomyosin 5.1 Insulin (bovine) 5.4 Fibrinogen (human) 5.8 g-Globulin (human) 6.6 Collagen 6.6 Myoglobin (horse) 7.0 Hemoglobin (human) 7.1 Ribonuclease A (bovine) 9.4 Cytochrome c (horse) 10.6 Histone (bovine) 10.8 Lysozyme (hen) 11.0 Salmine (salmon) 12.1
Protein crystals Page 133
Chromatographic separations Protein separation and purification by column chromatography From Lehninger Principles of Biochemistry
Column Chromatography: Ion Exchange From Lehninger Principles of Biochemistry
A schematic diagram illustrating the separation of several proteins by ion exchange chromatography using stepwise elution
Column Chromatograph: Size-exclusion Gel filtration chromatography can be used to estimate molecular masses From Lehninger Principles of Biochemistry
Hydrophobic interaction chromatography: Proteins contain hydrophobic amino acid side-chains, some of which are exposed at the surface of the protein. Proteins will therefore often bind to other hydrophobic molecules. Hydrophobic interaction columns are produced by covalently attaching hydrophobic molecules such as acyl chains or phenyl groups to insoluble carbohydrate resins.
Protein separation and characterization by Electrophoresis Migration of ions in an electric field is widely used for the analytical separation of biomolecules.
Polymerization of acrylamide and N,N¢-methylenebisacrylamide to form a cross-linked polyacrylamide gel Page146
SDS binds to proteins in amounts roughly proportional to molecular weight. • The high negative charge of the SDS-protein complex is greatly in excess of the inherent charge of the protein. • Electrophoresis in the presence of SDS therefore separates almost exclusively by molecular weight.
Polyacrylamide gel • Stain proteins to visualize, e.g., Coomassie blue (see page 93) • Rate of migration depends roughly on charge-to-mass ratio. • (Protein shape may also influence migration rate.) Larger proteins move more slowly Smaller proteins move faster From Lehninger Principles of Biochemistry
From Lehninger Principles of Biochemistry
A logarithmic plot of the molecular masses of 37 different polypeptide chains ranging from 11 to 77 kD versus their relative electrophoretic mobilities on an SDS-polyacrylamide gel
Setting up a purification Table Total Enzyme Activity: the activity per ml of enzyme solution multiplied by the total volume of that fraction. Total Protein: the protein concentration per ml of solution multiplied by the total volume of that fraction. Specific activity: Total Activity/Total Protein or Activity/Protein concentration Fold-purification: (Specific activity at a given step)/(Specific activity of starting sample) Yield = (Total activity at a given step) / (Total activity of starting sample)*100
Setting Up a Purification Table: Total Enzyme Activity: the activity per ml of enzyme solution multiplied by the total volume of that fraction. (If you have 50 ml of the original homogenate, the volume is 50 ml) Total Protein: the protein concentration per ml of solution multiplied by the total volume of that fraction. Specific activity: Total Activity/Total Protein or Activity/Protein concentration Fold-purification: (Specific activity at a given step)/(Specific activity of starting sample) Yield: (Total activity at a given step)/(Total activity of starting sample)*100
From Lehninger Principles of Biochemistry