Lecture 5 Protein purification techniques I

Question 1

How do we select a protein for isolation and purification?

Answer 1

• If possible find a source that contains large amounts
  of the desired protein
• If possible find a source that allows easy extraction
  and purification of the desired protein
• These aims are seldom possible

Question 2

How do we make a protein accessible for isolation/purification ?

Answer 2

• The protein should be in solution
• It is already accessible if the desired protein is in the
  extra-cellular medium
• The cell must be broken (a process called lysis) if the protein is located in the intra-cellular medium

Question 3

Why will proteins in a solution denature?

Answer 3

• The proteins in solution will denature unless they are
  stabilised against:
• Changes in pH ==> Add a buffer to the mixture
• Changes in Temperature ==> Cool the mixture to
  ~ 4°C
• Protease degradation ==> Add protease inhibitors

Question 4

How does protein isolation and purification operate?

Answer 4

Protein isolation and purification uses differences in the chemical and physical properties of proteins

Question 5

What differences in properties does protein separation use?

Answer 5

• Protein separation use differences in such properties as:
• solubility
• size
• charge
• Specific Binding Affinity

+ Usually, more than one step is required to separate
proteins completely one from another

+ After each step, a test - (an assay) - must be performed to see if the desired protein has been purified from others

Question 6

What are some protein isolation techniques?

Answer 6

• Salting Out
• Dialysis
• Gel-filtration chromatography
• Ultracentrifugation

Question 7

Describe the technique salting out.

Answer 7

• Salting out uses the solubility differences of proteins
• The solubility of proteins changes with the addition of ionic salts
• increasing salt concentration concentration decreases protein solubility
• Different proteins will precipitate out at different salt concentrations
• This results only in partial separation of the proteins NOT A FULL SEPARATION

Question 8

Describe the technique dialysis.

Answer 8

• Dialysis uses the size difference between proteins and small molecules to separate them
• Dialysis uses a semipermeable membrane
• Protein molecules are retained in the dialysis bag
• Small molecules pass through the bag into the external solution
• Ionic salts and buffering agents can be removed
• Dialysis can exchange one buffer for another
• This permits changes to be made in the pH of the protein solution

Question 9

Explain the technique gel-filtration chromatography.

Answer 9

• Gel-Filtration Chromatography uses the differences in the sizes of proteins
• This technique uses a column of porous beads
• The beads are formed from of a highly hydrated polymer gel

Examples of polymers used: Dextran (A polycarbohydrate), Agarose (Also a polycarbohydrate) and Polyacrylamide

• The protein mixture is placed onto the top of a column
• The protein mixture will flow down through the column and will be collected as fractions
• BUT the proteins in the mixture will have a range of sizes
• Smaller proteins can permeate the pores in the beads
• The flow of the smaller proteins is slowed by the beads
• Larger proteins cannot enter the beads
• The larger proteins flow faster around the beads in the column
• The larger proteins will be first through the column

Summarise:
Biggest proteins first through Beads (BfB)
-Large quantitiea of proteins can be separated but the separation is not well resolved unless there is a big difference between the sizes of the proteins

Question 10

Describe the technique Ultracentrifugation.

Answer 10

• Ultracentrifugation ultimately uses differences in the
  masses of proteins
• Ultracentrifugation employs very high rotation speeds
• Example: 75,000 rpm
  -Proteins are separated according to their sedimentation coefficient
• The sedimentation coefficient is proportional to protein mass
• A heavier protein will sediment down faster in an ultracentrifuge
• Buoyancy acts against the centrifugal force
• Related to protein density

One type of Ultracentrifugation: Zonal Centrifugation (also called Band Centrifugation or Gradient Centrifugation)

• This technique requires a density gradient inside the
  centrifuge tube
• The density gradient suppresses convection currents inside the tube
• These would prevent effective protein separation
• A density gradient can be produced by the mixing of low- and high-density solutions
• A typical example: 5% sucrose and 20% sucrose
• The protein mixture is placed on the density gradient in the centrifuge tube
  -Ultracentrifugation separates the proteins into bands
• These proteins can then be collected as fractions of the solution