Lecture 7 Chromatography Protein Purification

Question 1

Chromatography protein purification - why?

Answer 1

Allows bioactivity of proteins to be determined

Purified proteins are useful reagents for analysis (e.g. diagnostics) in biosynthesis and pharmaceuticals

Question 2

Two chromatographic separation techniques for proteins

Answer 2

1) by size : size exclusion aka gel filtration

2) by charge: ion exchange

Question 3

Extraction

Answer 3

An extraction of the protein to be purified must be made soluble

Easy for soluble globular proteins but difficult for membrane/structural/storage proteins which are hard to make soluble without denaturation

Differential centrifugation can be used to separate different cellular fractions based on size of cellular structures and proteins.

Precautions: minimise protein denaturation/degradation by protease enzymes in tissue by using buffered solutions and low temp.

Question 4

Radical centrifugal force

Answer 4

“Radial centrifugal force”
RCF=1.12rmm (Nrpm)²

Measured in “g” units and does not depend on centrifuge used

rmm= rotational radius measured in mm

Nrpm=rotational speed measured in revolutions per minute (RPM)

RPM does depend on centrifuge used so RCF preferred in report writing

Question 5

Solubility fractionation - simple purification by heat/salt/solvent

Answer 5

Quick initial purification of proteins can be made based on solubility.

Solubility at diff. pH or elevated temp (but beware of denaturation)

Solubility at v. low salt conc. - basis for differentiation of
serum albumins (soluble in water)
and
globulins (insoluble in water, soluble in dilute salt solution)

Solubility at high salt conc. - salting out. Carried out using high conc. Ammonium sulfate (minimises denaturation) diff proteins have diff solubility

Solubilties in mixtures of miscible org. solvents (beware denaturation)

• limited purification but useful

Question 6

Gel filtration

Answer 6

Protein separated by size of whole molecule.
Comparison of size of “native” protein compared to size of polypeptides that make it up can be used to work out how many polypeptides are present per molecule

E.g. Hb
polypep molecular weight= 16000 “native” protein molecule wt.=66000
Therefore there are 4 polypeptides per native protein molecule

Works best with relatively small quantities of proteins

Question 7

Ion exchange chromatography

Answer 7

Proteins separated by overall charge on molecule

Beads w/diff substituents either pos or neg charged at bio pH are available.

pH must be kept constant using a buffer but can be manipulated to alter separations

Works well with large quantities of proteins

Conditions required to elute specific proteins from column depends on charge carried by protein at the pH of the experiment
The higher the charge on the protein the tighter the binding and the higher the salt conc. required for elution

Question 8

Affinity chromatography

Answer 8

E.g. glucose binding protein separated from a complex mix by binding in a column where beads have a glucose residue covalently attached to form an affinity matrix.
Versatile technique but usually done on small scale due to difficulty and cost of preparing affinity matrices
Another affinity method is His-tagging

Question 9

Purifying proteins

Answer 9

Requires multiple steps carried out in succession.

Typical purification scheme below - note early stages use large scale techniques and later stages use processes for smaller quantities

Homogenisation
Salt fractionation
Ion exchange chromatography
Gel-filtration chromatography
Affinity chromatography