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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

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slide1

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.

slide2

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
slide3

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)
slide4

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.

slide5

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

slide6

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

slide7
Solubility of carboxyhemoglobin at its isoelectric point as a function of ionic strength and ion type

Page 131

slide10

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

slide12

Chromatographic separations

Protein separation and purification by column

chromatography

From Lehninger

Principles of Biochemistry

slide13

Column Chromatography:

Ion Exchange

From Lehninger

Principles of Biochemistry

slide15

A schematic diagram illustrating the separation of several proteins by ion exchange chromatography using stepwise elution

slide16

Column

Chromatograph:

Size-exclusion

Gel filtration chromatography can be used to estimate molecular masses

From Lehninger

Principles of Biochemistry

slide19

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.

slide21

Protein separation and characterization by

Electrophoresis

Migration of ions in an electric field is widely used for the analytical separation of biomolecules.

slide22

Polymerization of acrylamide and N,N¢-methylenebisacrylamide to form a cross-linked polyacrylamide gel

Page146

slide23

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.
slide24

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

slide25

From Lehninger

Principles of Biochemistry

slide26

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
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

slide29

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

slide30

From Lehninger

Principles of Biochemistry