Protein Purification

1
Protein Purification

• Molecular weight
• Charge
• Solubility
• Affinity

2
Molecular Weight

• Ultracentrifugation
• Dialysis
• Gel filtration
• SDS PAGE

3
Molecular Weight

• The lab in week 6 and 7 will involve separating a
  protein mixture by molecular weight using 2
  methods gel filtration and SDS PAGE
• Gel filtration separates by the native molecular
  weight
• SDS PAGE separates by the subunit molecular weight

4
Gel Filtration

• This method relies on a column of beads of a
  specified pore size. This is known as a molecular
  sieve.
• Proteins (and other macromolecules) above a
  certain cut-off size cannot fit into the pores
  and so migrate down the outside of the beads.
  They will elute first.
• Smaller molecules below the cut-off can permeate
  the pores and so take longer to travel down the
  column.

5
An elution profile

• Gel-filtration of the protein mixture. 1.2 ml of
  protein mixture (10 mg/ml) was loaded onto a 25
  cm X 2.5 cm diam. Sephadex G-50 column
  equilibrated with buffer (50 mM Tris HCl, pH
  7.5). The column was eluted with buffer at 1
  ml/min, collecting 2.5 ml fractions. The
  absorbance of each fraction was measured at 280
  nm and 410 nm.

Lighter
Heavier
6
SDS PAGE

• This technique involves loading a sample of your
  mixture onto a polyacrylamide gel (PAGE).
  Polyacrylamide works like agarose except the
  matrix has smaller pores and so polyacrylamide
  gels separate smaller molecules (like proteins).
  Agarose is used for much larger molecules such as
  DNA and RNA.

7
SDS PAGE

• Unlike DNA and RNA proteins do not have a nice
  constant charge to mass ratio and can have any
  charge at a given pH, depending on their
  sequence, hence pI.
• To overcome this problem proteins are coated with
  a detergent, SDS, which makes them negatively
  charged.
• They then separate by molecular weight.

8
SDS PAGE

• They then separate by molecular weight.
• The SDS will disrupt the secondary, tertiary and
  quaternary structure so the subunits will
  separate. For this reason SDS-PAGE separates by
  subunit molecular weight.

9
SDS-PAGE

1. Catalase, cytochrome C, a-lactalbumin
2. Hemoglobin, Cytochrome C, a-lactalbumin
3. BSA, cytochrome C, a-lactalbumin
4. Hemoglobin, myoglobin, a-lactalbumin
5. Ferritin, cytochrome C, a-lactalbumin
6. Ferritin, myoglobin, a-lactalbumin

1
2
4
3
5
6
lighter
10
Your Task

• Each pair will be given a mixture of three
  proteins. This mixture will be unique to your
  group. Your mixture will contain between 4 and 8
  mg of any three of the following proteins
  Myoglobin, Haemoglobin, Cytochrome c,
  a-lactalbumin, Ribonuclease, Bovine Serum
  Albumin, Ferritin and Catalase. It is your task
  to separate and identify these three proteins.

11
Clues to help you

• Your mixture of three proteins will either
  contain 2 heavy and 1 light protein or 1 heavy
  and 2 light proteins. For the purposes of this
  experiment, a heavy protein is defined as one
  with a molecular weight of over 50,000 and a
  light protein is one with a molecular weight of
  less than 50,000. The two smaller/heavier
  proteins in the mixture have pIs that differ by
  at least 2 pH units. These can be separated by
  ion exchange chromatography at pH 7.5.

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Hint..

• If you can separate 2 proteins by ion exchange
  chromatography at pH 7.5 then the 2 proteins must
  have pIs on either side of 7.5 so they are
  opposite charges at PpH 7.5.
• Knowing information about the possible proteins
  at the back of the notes for this lab session
  what can you conclude before coming to class?

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Charge

• Ion Exchange Chromatography
• Native gel electrophoresis
• Isoelectric focusing

14
Charges on proteins

• Different proteins have different native charges.
• The overall charge on a protein will depend on
• The sequence
• The pH

15
Determining the pI of a protein

• It can be predicted from the difference between
  the sum of the acidic side chains (asp glu) and
  the sum of the basic side chains (lys arg
  his).
• It is determined experimentally by techniques
  such as isoelectric focusing. The protein is
  placed in a pH gradient and subjected to an
  electric field. The protein moves to its pI.

16
Determining the pI of a protein

• Those proteins with more acidic residues will
  have a lower pI
• Those proteins with more basic residues will have
  a higher pI.

17
Estimating the charge of a protein

• What we really want to know is the charge of a
  protein at a particular pH, like 7.
• How do we use pI data to predict the charge of
  our protein?
• Acidic residues lower the pI
• Basic residues raise the pI.

18
Estimating the charge of a protein
pI 5
H
OH-
OH-
H
Protein becomes increasingly ve
Protein becomes increasingly -ve
19
Estimating the charge of a protein
At pH 3 the protein will be ve
pH 3
pI 5
H
OH-
OH-
H
Protein becomes increasingly ve
Protein becomes increasingly -ve
20
Estimating the charge of a protein
At pH 7 the protein will be -ve
pI 5
H
pH 7
OH-
OH-
H
Protein becomes increasingly ve
Protein becomes increasingly -ve
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At a particular pH..

• If the pH of the environment is below (more
  acidic gtH) the pI then the protein will be
  positive (ve)
• If the pH of the environment is above (more basic
  gtOH-) the pI then the protein will be negative
  (-ve).

22
Ion Exchange Chromatography

• If the column is positively charged i.e. DEAE
  then.
• Proteins with pIs l lt the pH of the buffer will
  be negatively charged and bind to the column.
• Proteins with pIs gt the pH of the buffer will be
  positively charged and will not bind to the
  column but elute.

23
Ion Exchange Chromatography

• If the column is negatively charged charged i.e.
  carboxymethyl then.
• Proteins with pIs l lt the pH of the buffer will
  be negatively charged and not bind to the column
  but elute.
• Proteins with pIs gt the pH of the buffer will be
  positively charged and will bind to the column.

24
Native Gel Electrophoresis

• Proteins with pIs l lt the pH of the buffer will
  be negatively charged and will move to the anode
  (ve), the red electrode!!
• Proteins with pIs gt the pH of the buffer will be
  positively charged and will move to the cathode
  (-ve), the black electrode!!

25
Isoelectric Focusing

• A pH gradient is set up along the length of the
  gel
• An electric field is applied
• Proteins move to the point where they no longer
  have a charge i.e. their pI
• Used as the first dimension of 2D gel
  electrophoresis

26
Proteomics

• A combination of isoelectric focusing (first
  dimension) and SDS PAGE (second dimension) can
  separate the complete proteome of a cell!
• You produce spots which can be cut out and
  analysed by mass spectrometry.
• Compare to libraries of proteins

27
2D gel electrophoresis
28
Affinity

• Exploited with cloning
• His-tagged proteins purified on Nickel columns.
• GST fusion proteins purified on glutathione
  columns.