Lecture Notes: Enzyme Sens and Technology

Overview

• The lecture focuses on enzyme properties and production, specifically gene isolation, cloning, and protein purification.
• Different systems for protein production: bacterial, yeast, bacteriophage, insect, and mammalian.

Gene Isolation and Cloning

• Approaches for isolating the gene of interest:
  • Genomic library
  • cDNA library
  • Site-specific primers
• Cloning vectors use restriction enzymes and ligase enzymes to clone genes into hosts (bacterial or eukaryotic).

Protein Purification Techniques

• Methods for isolating proteins and purifying enzymes:
  • Cell disruption methods
  • Chromatography principles

Chromatography Techniques

1. Ion Exchange Chromatography (IEC)

   • Exploits charge differences (positive or negative) on proteins.
   • Separation based on charge interaction with a charged matrix.
   • Types:
     • Cation Exchange: Negatively charged matrix attracts positively charged proteins.
     • Anion Exchange: Positively charged matrix attracts negatively charged proteins.
   • Operational Steps:
     • Select and prepare matrix
     • Run column with mobile phase
     • Load sample and elute with salt gradients
     • Column regeneration with high ionic strength solution

2. Hydrophobic Interaction Chromatography (HIC)

   • Utilizes hydrophobic patches on proteins.
   • Salting-in and salting-out effects:
     • Low salt increases solubility (salting-in).
     • High salt exposes hydrophobic patches, leading to binding (salting-out).
   • Operational Steps:
     • Equilibrate column in high salt solution
     • Bind proteins by exposing hydrophobic patches
     • Elute by decreasing salt concentration or changing mobile phase polarity
     • Column regeneration with urea or detergents

Protein Properties and Chromatography

• Protein folding and structure:
  • Hydrophobic core and polar periphery.
• Proteins have unique surface areas, charges, and hydrophobic patches exploitable by chromatography.
  • Ion Exchange: Based on charge differences.
  • Hydrophobic Interaction: Based on hydrophobic patches.

Important Factors

• pH and ionic strength significantly influence chromatography outcomes.
• Protein's pi value critical for selecting chromatography method:
  • Below pi: Cation exchange suitable.
  • Above pi: Anion exchange suitable.

Conclusion

• Understanding protein properties and selecting appropriate chromatography techniques are crucial for effective protein purification.
• Future lectures will cover more on gel filtration and affinity chromatography.