Protein Purification

Why purify a protein?

• To study its function
• To analyze its physical properties
• To determine its sequence
• For industrial or therapeutic applications

Steps in Recombinant Protein Purification:

1. Design expression plasmid, transform, select
2. Grow culture of positive clone, induce expression
3. Lyse cells
4. Isolate and Purify the protein
5. Assess purity on SDS-PAGE

Lysis of the Cell

Chemical:

      alkali, organic solvents, detergents

Enzymatic:

      lysozyme, glucanases, chitinase

Physical:

      osmotic shock, freeze/thaw

Mechanical:

       Sonication, homogenization, wet milling, French press

Purification techniques

1.  based on molecular size

  -  dialysis and ultrafiltration

  -  density gradient centrifugation

  -  size-exclusion chromatography)

2.  based on solubility of proteins

  -  isoelectric precipitation

  -  salting out

3.  based on electric charge

  -  ion-exchange chromatography

  -  electrophoresis