L3: Protein Purification

Question 1

First steps of protein purification

Answer 1

1. For intracellular proteins, cells need to be lysed first.
   • Insect and mammalian cells are lysed by a homogeniser or detergent.
   • E. coli can be lysed by lysosome to degrade cell wall, pressure drop/french drop (good for large scale), sonication (heating can damage proteins, not scalable), Osmotic shock for periplasm
2. Secreted - dialysis, ultrafiltration

Question 2

Bulk fractionation

Answer 2

Low resolution protein purification technique.

1. Cell lysis -> differential centrifugation
2. Secreted protein -> dialysis/ultrafiltration

Question 3

In centrifugation, sedimentation rate is dependent on the _____ of particle

Answer 3

density, size, shape, solution density

Question 4

Ultrafiltration

Answer 4

• Low resolution bulk fractionation technique.
• Membrane filtration where pressure is applied to force solvent and small molecules through the membrane, concentrating the larger macromolecules
• Batch or continuous
• Concentration of macromolecules
• Desalting
• Fractionation