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Topic : Recombinant Protein Production

GS-3 Mains  : Science and Technology

Revision Notes

What are recombinant proteins?

• Proteins made using genetically modified organisms (bacteria, yeast, or mammalian cells).
• Used in various medical applications like vaccines, insulin, and monoclonal antibodies.
• Produced by inserting the gene coding for the desired protein into a host cell.

Why is yeast (Pichia pastoris) preferred?

• Most widely used organism for recombinant protein production.

Traditional method (methanol-induced):

• Uses a promoter activated by methanol to induce protein production.
• Challenges:
  • Safety hazard: Methanol is highly flammable and toxic.
  • Oxidative stress: Byproduct of methanol metabolism damages cells and proteins.

Novel method (MSG-induced):

• Uses monosodium glutamate (MSG), a safe food additive, to activate a different promoter.
• Advantages:
  • Safer: MSG is non-flammable and easier to handle.
  • More efficient: Eliminates oxidative stress from methanol.

Applications of the new method:

• Mass production of valuable proteins in biotech industries.
• Products: Milk and egg proteins, baby food supplements, nutraceuticals, and therapeutic molecules.
• Scalable for widespread adoption due to safety and efficiency.
• Significant advancement in biotechnology for a more sustainable and safer approach.

Recombinant protein production process:

1. Gene Cloning:
   • Isolate the gene coding for the protein of interest.
   • Insert the gene into a vector (plasmid) for transfer into host cells.
   • Introduce the vector into host cells (bacteria, yeast, insect, or mammalian).
2. Expression Systems:
   • Prokaryotic (e.g., E. coli): Simple, fast, and cost-effective for non-glycosylated proteins.
   • Eukaryotic:
     • Yeast (e.g., Saccharomyces cerevisiae): Can perform some post-translational modifications.
     • Insect Cells (e.g., Baculovirus system): High yield and proper folding for complex proteins.
     • Mammalian Cells (e.g., CHO cells): Best for complex proteins with human-like modifications.
3. Protein Purification:
   • Release the protein from host cells through cell lysis.
   • Purify the target protein using chromatography techniques:
     • Affinity chromatography (specific ligand)
     • Ion exchange chromatography (protein charge)
     • Size exclusion chromatography (protein size)
4. Protein Characterization:
   • SDS-PAGE: Determines protein molecular weight.
   • Western Blot: Confirms protein presence using specific antibodies.
   • Mass Spectrometry: Provides detailed protein mass and structure information.
   • Activity Assays: Assesses the functional activity of the protein.

Applications of recombinant proteins:

• Therapeutics: Insulin, erythropoietin, monoclonal antibodies.
• Vaccines: Hepatitis B, HPV.
• Diagnostics: Enzymes and antigens used in assays.

Advantages of recombinant protein production:

• High yield of proteins.
• Highly purified proteins.
• Consistent production across batches.

Challenges:

• Not all post-translational modifications can be done in prokaryotic systems.
• Solubility issues: Some proteins form insoluble aggregates.
• Cost: Eukaryotic systems can be expensive.