Lecture 4 Protein purification

Question 1

What are reasons to purify a protein?

Answer 1

Investigate a protein without “contamination” Utilize a protein for biotechnology/medicine (often large scale)

Question 2

Protein purification: strategy. What are important steps?

Answer 2

• Choice of protein source (bacteria, plant, yeast, human, etc..)
• Disrupt cells/solubilize protein (osmotic shock, detergent…)
• Stabilization of protein
• Purifications step(s)
• Assay of protein (activity, immunological etc..)

Question 3

Proteins are very unstable. What are things to keep in mind when handling them with care?

Answer 3

Stabilization against:

• Extreme pH: add buffer to protein solution
• Extreme temperature: keep cool (<20oC often 4oC) maximum
• Proteolysis: keep cool, add protease inhibitors
• Hydrophobic environment: keep solution surface small, protein concentration high
• Bacteria: add bactericidal compound (e.g. sodium azide)

Question 4

What is Column-Chromatography?

Answer 4

Separating components of a protein mixture using a column of porous material (Beads ~50 mM in size)

Question 5

Name 3 kinds of chromatography and on the basis of what are proteins seperated?

Answer 5

Proteins will be separated according to: Charge (Ion exchange chromatography) Size (Size exclusion chromatography) Affinity for particular surface (affinity chromatography)

Question 6

Ion-exchange chromatography: what happens here? How can you retard only - / + charged proteins?

Answer 6

Ion-exchangechromatography:chargedproteins retarded by column- charged proteins =>Anion exchange+ charged proteins=>cation exchange

Question 7

Size exclusion (gel filtration) chromatography: what happens?

Answer 7

Size exclusion (gel filtration)Chromatography:Small proteins can enter pores and are retarded by column material surface

Question 8

Affinity chromatography: what happens? What type of proteins is it often used for? Example?

Answer 8

Specific ligand is bound to column materialOnly proteins with specific binding motifretardedOften used for recombinant proteinsExample: Ni-NTA column

Question 9

What is a frequently used parameter to assess intactness?

Answer 9

Frequently used parameter to assess intactness: specific activity (micromol substrate * mg protein * min)