Protein Purification

❓ Why Purify Proteins?

• To determine structure and mechanism of action

• Proteins are separated based on:

  • Charge

  • Size

  • Affinity

  • Hydrophobicity

🧪 General Workflow

1. Cell lysis (extract protein)

2. Clarify lysate (remove debris)

3. Initial fractionation (e.g. salting out, centrifugation)

4. Chromatography steps (1–3 methods)

5. Final polishing (often size exclusion)

6. Concentration + storage

🎯 Goal: Maximum purity, minimum steps/resources.

🌀 Ultracentrifugation

🔬 Analytical

• Sedimentation velocity → shape, mass, interactions

• Sedimentation equilibrium → mass only

⚙ Preparative

• Gradient separations:

  • CsCl → nucleic acids

  • Sucrose → organelles & membranes

⚡ Electrophoresis

• Native PAGE → separates by charge:mass

• SDS-PAGE → separates by mass only
  (SDS coats protein with uniform negative charge)

• 2D-PAGE → 1st by charge (IEF), 2nd by mass (SDS-PAGE)

🧂 Salting Out (Ammonium Sulphate Precipitation)

1. Polar residues interact with water

2. Salt competes for water

3. Water is pulled away → protein aggregates

4. Centrifuge:

   • Keep pellet/supernatant containing target protein

🧪 Chromatography Methods

🧬 Key Terms

• Stationary phase: Resin or matrix

• Mobile phase: Flowing liquid (buffer)

• Elute: Wash bound protein off the column

• Eluate: The collected sample

🧼 Hydrophobic Interaction Chromatography (HIC)

• Uses phenyl/octyl Sepharose

• Salt promotes binding (unlike other methods)

• Workflow:

  1. Equilibrate with high salt buffer

  2. Load sample

  3. Wash out impurities

  4. Elute with decreasing salt gradient

  5. Fractionate & monitor at A280

🔳 Size Exclusion Chromatography (SEC / Gel Filtration)

• Separates by size

• Isocratic elution (no gradient)

• Larger proteins elute first

• Smaller proteins elute last

• Use to:

  • Purify

  • Desalt

  • Buffer exchange

  • Estimate molecular weight

📊 Elution Volumes

• V₀: Void volume (excluded molecules)

• Vt: Total liquid volume

• Vc: Column volume (geometric)

• Pore volume = Vc - V₀

⚖ Ion Exchange Chromatography (IEX)

• Separate by charge

🧪 Principle

• Media contains charged groups

• Opposite charges bind, same charges elute

🧬 Isoelectric Point (pI)

• pI = pH where net charge = 0

  • pH > pI → protein is negative

  • pH < pI → protein is positive

➕ Cation Exchange

• Negative resin (e.g. CM-cellulose, S-Sepharose)

• Done at pH < pI

➖ Anion Exchange

• Positive resin (e.g. DEAE-cellulose, Q-Sepharose)

• Done at pH > pI

🧲 Affinity Chromatography

🔗 Principle

• Specific binding between protein and immobilised ligand

💡 Types

1. Epitope tags (e.g. FLAG, Myc, HA)

2. Engineered tags (e.g. 6xHis, GST, MBP)

3. Native interactions (e.g. NAD(P)H, heparin binding)

🧬 Tagging

• N-/C-terminal fusion

• Often with protease cleavage site

🧲 IMAC (Immobilised Metal Affinity Chromatography)

• Uses His-tag (usually 6 residues)

• Ni-NTA or Co²⁺ resin

🧪 Workflow

1. Bind in low imidazole

2. Wash in low imidazole

3. Elute with high imidazole

🧠 Add imidazole to avoid non-specific binding

💡 Other Affinity Systems

🧬 Lysozyme

• Enzyme that cleaves peptidoglycan

• Acts on 1,4 linkage between NAM and NAG

• Found in tears, milk, saliva

• Positively charged surface

🧫 Membrane Protein Purification

• Proteins must be kept soluble

• Aggregation risk in polar environments

🧴 Detergents

• Form micelles around hydrophobic proteins

• CMC = critical micelle concentration

• CMT = critical micelle temperature

• Aggregation number = molecules per micelle

🧠 Rapid Recap

SEC → Size
IEX → Charge (pI & pH crucial!)
HIC → Hydrophobicity (high → low salt elution)
Affinity → Specific binding (tags or native ligands)
Ultracentrifugation → Mass & shape (analytical/prep)
Salting Out → Competitive water removal → aggregation